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VOL. 38, NO. 1-2 1996
MALACOLOGIA
International Journal of Malacolog y
Revista Internacional de Malacologia
Journal International de Malacologie
Международный Журнал Малакологии
Internationale Malakologische Zeitschrift
Vol.
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2 Dec.
8 Jan.
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1988
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1995
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. 1996
VOL. 38, NO. 1-2 MALACOLOGIA
CONTENTS
FADWA A. ATTIGA & HAMEED A. AL-HAJJ
Ultrastructural Study of Euspermiogenesis in Clypeomorus Bifasciata and Clypeo-
morus Tuberculatus (Prosobranchia: Cerithiidae) With Emphasis on Acrosome
Éoimationme ee ооо оо оо осо асов особо ove creer
RÜDIGER BIELER & RICHARD Е. PETIT
Additional Notes on Nomina First Introduced by Tetsuaki Kira in “Coloured Illustra-
tions of the: ShellSiOf Japan? se soccer ee ce вое о еее
M. E. CHASE & R. C. BAILEY
Recruitment of Dreissena Polymorpha: Does the Presence and Density of Conspe-
cifics Determine the Recruitment Density and Pattern in a Population? ...........
KENNETH C. EMBERTON
Microsculptures of Convergent and Divergent Polygyrid Land-Snail Shells ........
KENNETH C. EMBERTON, TIMOTHY A. PEARCE & ROGER RANDALANA
Quantitatively Sampling Land-Snail Species Richness in Madagascan Rainforests .
MARIA FERNANDA LOPEZ ARMENGOL
Taxonomic Revision of Potamolithus Agapetus Pilsbry, 1911, and Potamolithus
Buschii (Frauenfeld, 1865) (Gastropoda: Hydrobiidae) ..........................
MARTIN HAASE & ERHARD WAWRA
The Genital System of Acochlidium fijiense (Opisthobranchia: Acochlidioidea) and its
Inferred\EUNCtiOn Re den Me ire mer ser coca error ve
WALTER R. HOEH & MARK E. GORDON
Criteria for the Determination of Taxonomic Boundaries in Freshwater Unionoids
(Bivalvia: Unionoida): Comments on Stiven and Alderman (1992) ................
С. M. KUCHENMEISTER, D. J. PRIOR & I. G. WELSFORD
Quantification of the Development of the Cephalic Sac and Podocyst in the Terres-
tral@astropodilimax Maximus Eos oa во ea an as
RICHARD E. PETIT & RÜDIGER BIELER
On The New Names Introduced in the Various Printings of ‘‘Shells of the World in
Colour” [Vol. | by Tadashige Habe and Kiyoshi Ito; Vol. Il by Tadashige Habe and
Sadao: Kosudel za. 0m mais alcala soc dos ea aaa ae en
DR. F. D. POR & DR. R. M. POLYMENI
A Call for a New International Congress of Zoology ............................
PETER D. ROOPNARINE
Systematics, Biogeography and Extinction of Chionine Bivalves (Bivalvia: Veneridae)
in Tropical America: Early Oligocene-Recent ..................................
LUIZ RICARDO L. SIMONE
Anatomy and Systematics of Buccinanops Gradatus (Deshayes, 1844) and Bucci-
nanops Moniliferus (Kiener, 1834) (Neogastropoda, Muricoidea) From the Southeast-
enn Coast of Brazilian dite oasis iO buste
CHRISTINA M. SPOLSKY, GEORGE M. DAVIS & ZHANG YI
Sequencing Methodology and Phylogenetic Analysis: Cytochrome b Gene Sequence
Reveals Significant Diversity in Chinese Populations of Oncomelania (Gastropoda:
POMATOPSIAAE) ола ев ea
P. TATTERSFIELD
Local Patterns of Land Snail Diversity in a Kenyan Rain Forest ..................
LAURA R. WHITE, BRUCE A. MCPHERON, & JAY R. STAUFFER, JR.
Molecular Genetic Identification Tools for the Unionids of French Creek, Pennsylva-
A ie O E OO SO
DAZHONG XU & MICHELE G. WHEATLY
CA Regulation in the Freshwater Bivalve Anodonta Imbecilis: |. Effect of Environmen-
tal CA Concentration and Body Mass on Unidirectional and Net CA Fluxes ......
1996
47
33
143
223
153
35
229
103
87
213
161
181
59
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MALACOLOGIA, 1996, 38(1-2): 1-17
TAXONOMIC REVISION OF POTAMOLITHUS AGAPETUS PILSBRY, 1911, AND
POTAMOLITHUS BUSCHII (FRAUENFELD, 1865) (GASTROPODA: HYDROBIIDAE)
María Fernanda López Armengol
Instituto de Embriología, Biología e Histología, Facultad de Ciencias Médicas-CONICET,
Universidad Nacional de La Plata, Calle 60 y 120 (1900), La Plata, Argentina
ABSTRACT
Potamolithus agapetus Pilsbry, 1911, and P. buschii (Frauenfeld, 1865) are related species
that live sympatrically in Río de la Plata.
Studies carried out on populations of both species from Río de la Plata show that P. agapetus
presents a marked secondary sexual dimorphism on shell shape and size.
The female shell is bigger than male shell, and its body whorl shape is subglobose, with a
rounded angle at the basal periphery and another angle a short distance below the suture. The
male body whorl shape is usually rounded without keels and seldom with a round angle at the
basal periphery.
Females of P. agapetus are very similar to the shell of P. buschii, which lacks secondary
sexual dimorphism. For that reason, P. agapetus females were excluded from the original
description by Pilsbry (1911), and seemingly included in subsequent enlarged descriptions of
Р. buschii.
Both species share the same body whorl shape, but both present different degrees between
angulose to globular shapes. They can be distinguished by shell color pattern, columella width,
body whorl sculpture, head pigment pattern, eyebrow position, nuchal node size in females, gill
filament number and range, and in the shape and number of cusps on the central and lateral
radular teeth.
Key words: Potamolithus, Hydrobiidae, taxonomy, sexual dimorphism.
INTRODUCTION
The genus Potamolithus comprises small
(up to 7 mm long), thick-shelled gastropods
that inhabit rivers and streams (Pilsbry, 1911;
López Armengol, 1985).
This genus, exclusively South American
and endemic in Ribeira, Itajai-agu and Jacuhy
rivers in southern Brazil and Uruguay River,
part ofthe Paraná and Río de la Plata drainage
systems (López Armengol, 1985).
Controversial aspects of authorship and
type species (ICZN Case 2801; López Armen-
gol & Manceñido, 1992; Kabat, 1993; Kabat &
Hershler, 1993; Manceñido 8 López Armen-
gol, 1993) have been cleared up by ICZN ac-
tion (ICZN Opinion 1779, 1994) fixing Pilsbry
& Rush as the authors of this genus and Pot-
amolithus lapidum (d'Orbigny, 1835) as its
type species.
In 1911, Pilsbry presented a key to species
and subspecies and a description of the
known species which were arranged in four
groups. Parodiz (1965) gave a description of
Potamolithus species and added new char-
acters and geographical data. Davis & Pons
da Silva (1984) described the anatomy of P.
ribeirensis from Feitoría River, Brazil, and dis-
cussed phylogenetic relationships and con-
vergence with other hydrobiid and pomatiop-
sid genera.
The descriptions of Potamolithus species
were based on shell features, and only a few
specimens were studied in some cases.
Potamolithus agapetus Pilsbry, 1911, and
Potamolithus buschii (Frauenfeld, 1865) are
sympatric in Río de la Plata (Pilsbry, 1911;
López Armengol, 1985). According to Pilsbry
(1911) both species belong to the ‘group of P.
buschii”” because they share the same gen-
eral shell shape: both equally wide and high
with a normal length spire, a simple lip, and a
flattened columella. Juveniles of P. buschii are
not always readily distinguishable from imma-
ture P. agapetus. Potamolithus agapetus was
originally described as the smallest Potamo-
lithus known and has a globular-conic shell.
On the other hand, P. buschii was originally
described as having a wide and carinate shell.
Studies carried out on populations of both
species from Rio de la Plata show that spec-
imens with a shell morphology agreeing with
the original description of P. agapetus are all
males. On the other hand, a great variability
2 LÓPEZ ARMENGOL
FIG. 1. Scanning electron micrographs of the shell of P. agapetus. A, B, males; C, D, females. Body whorl
periphery: A, globose, and C, angular. F, lateral view: note the concave body whorl base. G, enlargement
of the shell showing the surface faintly marked with growth-lines and some pits. Scale bar A-E = 1 mm; F
= 50. Lim.
TWO RELATED SPECIES OF POTAMOLITHUS 3
B
==
FIG. 2. Variation in the shape of the shell in P. agapetus. A, females. B, males. Scale bar = 1 mm.
TABLE 1. Whorl number of P. agapetus. Frequency of males and females at each whorl stage present
at the localities studied. % = percentage of population.
Anchorena beach
Whorls d Q % g
eroded 7 15 3.12 2
2.50 — = — —
3.00 12 1 2.19 5
325 104 ПИ 20.44 7
3:50, 42 68 18.58 1
3375: 7 221 38.51 2
4.00 — 86 14.53 2
4.25 — 12 2.03 ee
N = 172 420 19
was observed in P. buschii in such charac-
ters as radula, head pigmentation, number of
gill filaments, and the size of the female
nuchal node.
The aim of this work is to redescribe both
P. agapetus and P. buschii.
MATERIALS AND METHODS
Localities studied were: Rio de la Plata,
Anchorena beach, Argentina (34°29’S,
58°28’W), col.: López Armengol, 30-IV-1984,
Colección Malacológica del Museo de La
Plata, MLP 4652; mouth of Rio San Juan
where it empties into Rio de la Plata, Uruguay
(33°17’S, 57°58’W), col.: Perez Duhalde, 15-
VII-1989, МЕР 4986; Rio de la Plata, Isla San
Gabriel, Uruguay (34°29’S, 57°52’W),col.: Ló-
Rio San Juan
Isla San Gabriel’s
? % 3 ? %
12 18.92 2 2 4.12
— — a 1 9.19
=> 6.76 18 7 25.77
= 9.46 19 1 20.62
1 2.70 й 6 13.40
qt WOW 2 20 22.68
29 41.89 — 8 8.25
2 2:10 — = —
95 92 45
pez Armengol-Casciotta, 17-11-1985, МЕР
4655.
The samples were taken randomly and in-
clude all individuals of all size classes at a
single site in the river. The sample for the
number of individuals and their sex for each
whorl number consisted of 592 specimens of
P. agapetus and 289 specimens of P. buschii.
These samples were drawn from an initial
population of 3,404 individuals (MLP 4652).
Specimens were measured by ocular mi-
crometer in a Wild M-5 stereoscopic micro-
scope. All specimens studied were unpara-
sitized. Measurements are those of Hershler
8 Landye (1988). The following ratios were
formed using some of this data: shell length/
body whorl length; body whorl length/shell
width; shell length/shell width and aperture
length/shell length.
TABLE 2. Shell measurements (m
to 3.75 whorls of P. agapetus (MLP 4652).
LÓPEZ ARMENGOL
significant difference between sexes, P< .001.
Characters
Shell length
Body whorl length
Spire length
Shell width
Aperture length
Aperture width
Columella width
Umbilical area width
Shell length/body whorl length
Body whorl length/shell width
Shell length/shell width
Aperture length/shell length
m) and ratios for 29 males and 44 females of 3.50
X + standard deviation (range). * =
Number of whorls was counted according
to Emberton (1985), but 0.25, 0.5 and 0.75
were the fractions considered. Body whorl
and penultimate whorl convexity were calcu-
lated following Hershler 8 Landye (1988).
Whorl convexity value is directly proportional
to whorl convexity.
Shells and radulae were studied and pho-
tographed using scanning electron micro-
scope (Jeol JSM-T 100). Heads were dried
by the critical point method.
The position and distance between the
base of penis or nuchal node with respect to
the lobes of the eyes and the angle of the
base of penis or nuchal node with respect to
the mid-line of the neck were calculated on
fixed material, following Davis et al. (1986).
Statistical analyses were limited to calcu-
lating the means, standard deviations, and
standard 't' test for sexual dimorphism in
shell measurements and ratios and gill fila-
ment number. The significance level ac-
cepted was Р < .001. Xi? was performed to
evaluate sex ratio = 1:1.
males females SD P < .001
2.32 + 0:25 2.88 + 0.26 5
(1.95 — 3.09) (2.27 — 3.24)
2.08 + 0.24 2.56 + 0.24 5
(1.76 2.77) (1.95 — 2.96)
0.25 + 0.04 0.32 + 0.05 =
(0.15 = 0:32) (0.19 — 0.44)
2.28 + 0.26 2:81 ==10.27 .
(1.95 — 3.09) (220321)
1.74 + 0.22 2.17 + 0.20 2
(1.45 — 2.39) (1.76 — 2.52)
1.22 + 0.16 510.15 *
(ОТ = 1.76) (1.20 — 1.83)
0.24 + 0.04 0.30 + 0.06 És
(0.16 — 0.32) (0.19 — 0.44)
0.18 + 0.07 0.22 + 0.08
(0.06 — 0.38) (0.06 — 0.38)
1.12+0.02 1.18 0:02
(1.08 — 1.18) (1.08 — 1.17)
0.91 + 0.03 0.91 + 0.04
(0.84 — 0.99) (0.83 — 0.98)
1.02 + 0.02 1.02 + 0.04
(0.92 — 1.09) (0.96 — 1.07)
0.73 + 0.04 0.75 + 0.04
(0.63 — 0.80) (0.63 — 0.83)
RESULTS
Potamolithus agapetus Pilsbry 1911
Potamolithus agapetus Pilsbry 1911: 578, pl.
40, fig. 10, 10a.
Potamolithus agapetus Parodiz 1965: 9
Type material: Academy of Natural Sciences
of Philadelphia 69,683.
Type locality: Río de la Plata, at Isla San
Gabriel, near Colonia, Colonia Department,
Uruguay.
Description
The shell is globose-conic to subglobose
(Figs. 1, 2) and solid but not thick. The color
is uniform light brown. The surface is rather
smooth, faintly marked with growth lines (Fig.
1F). The spire is 11% of the shell length. The
number of whorls is most frequently between
3.00 and 4.00 (Table 1), slightly convex
(penultimate whorl convexity = 0.20 and
body whorl convexity = 0.14) in outline. The
TWO RELATED SPECIES OF POTAMOLITHUS 5
A
475 A A
450
475 AAAA
5 400 А AAAAAA
2
3
© 375 e o. . 0 oAAAAAA, АА
©
о
5
=> o o Фо} оо} 1 { ora A
3.25 e bobrcoojtos A
300
20 3.0 4.0 50
Shell length (mm)
B
425 A a is
= 700 A 3444444444444
2
3
5 ars POVNET TE NN ET TEEN
©
pe]
Е
2 350
325
Shell length (mm)
FIG. 3. Scatter-diagram for the number of whorls and shell length. A, P. agapetus (82 males and 61
females). B, P. buschii (128 males and 108 females). Note the sexual dimorphism in shell size in P.
agapetus. Males (black circles), females (black triangles). One symbol may represents more than one
specimen.
6 LÓPEZ ARMENGOL
FIG. 4. Pigment patterns of P. agapetus. A, head-neck, dorsal view. B, penis, right side. Scale bar = 1 mm.
body whorl base is concave in dorsal view
(Fig. 1E). The aperture is oblique, inclined
about 35° to 42° (X = 39°) towards the coiling
axis, rounded-ovate, and angular at the top.
The columella is wide and flattened (Fig. 1B,
D).
Shells with discontinous peristome have a
thin outer lip, and the umbilical area can be
present or absent. When it present, is narrow
and bounded by an angle. In shells with con-
tinous peristome (Fig. 1B, D), the inner lip is
heavily calloused and the outer lip is simple
and thin. There is a rather conspicuous um-
bilical area bounded by an angle or an acute
ridge. Some specimens have an umbilical
opening.
There is sexual dimorphism in shape and
shell size. The shape of the body whorl in
males is usually globose (Figs. 1A, 2B). How-
ever, some males have a shell with a rounded
angle below the suture or with two angles,
one below the suture and the other at the
basal periphery (Fig. 2B). Males have a
rounded outer lip (Fig. 1B). The female body
whorl shape is usually subglobose, with two
rounded angles, one below the suture and
the other at the basal periphery (Figs. 1C,
2A); the outer lip is sharp (Fig. 1D).
The females are larger than males with the
same number of whorls (Fig. 3A). No sexual
dimorphism in umbilical area width and cal-
culated ratios were observed. Statistics on
shell dimensions for males and females of
3.50 to 3.75 number of whorls are given in
Table 2.
There was no significant difference in num-
ber of males and females at Anchorena
Beach (0.76:1).
The head can be unpigmented or with a
band of melanin in the snout, or with two
V-shaped bands orientated with the vertex
pointing from the snout to the neck. Another
band runs dorsally in the middle of each ten-
tacle. There is a concentration of white
spheric granules above and around the eyes
(“eyebrows””) (Fig. 4A), and eye lobes are
slight swellings at the base of each tentacle.
The neck of females bears a protuberance
called nuchal node (Davis & Pons da Silva,
1984). The position of the nuchal node base
is mainly to the right of the mid-line of the
head. The nuchal node is X = 0.25 mm + 0.02
(0.24-0.30) high (Fig. 5A, B). The distance be-
tween the base of the nuchal node and the
eyes is X = 0.31 mm + 0.09 (0.15-0.4). The
angle of the base of the nuchal node (with
respect to the mid-line of the neck) is X = 52°
(34°-72°).
The penis is simple, without appendages;
with a black spot at the distal end (Figs. 4B,
5C). The distance between the base of the
penis and the lobes of the eyes is X = 0.14
mm + 0.01 (0.12-0.15). The angle of the base
of the penis (with respect to the mid-line of
the neck) is X = 23° (14°-30°).
There are 19 to 28 gill filaments (Fig. 6),
TWO RELATED SPECIES OF POTAMOLITHUS 7
FIG. 5. Scanning electron micrographs of the
head-neck of P. agapetus. A, dorsal view of female
head-neck, showing the nuchal node. B, right side
of the head-neck of a female. C, left side of the
head-neck and fully erect penis of a male. Scale
bar = 200 um.
with no indication of sexual dimorphism in-
their number (X = 23.00 + 2.30 and 23.87 +
2.64 for males and females respectively).
Radula typically taenioglossate (Fig. 7), the
statistics and cusp formulae given in Table 3.
Distinctive features are: the concave hollow
in the middle of the anterior cusp of the cen-
tral teeth (Fig. 7C); the external edge of lateral
angle of the central teeth is sometimes
curved; the innermost pair of basal cusps
arise from the face of the tooth; there is a
concave hollow between the basal cusps and
basal process, and the basal process is
prominent.
There were no differences among the
blades of the lateral tooth (Fig. 7A, B, D, E).
There is a pronounced posterior projection
on the face of the lateral tooth (Fig. 7D).
Potamolithus buschii (Frauenfeld, 1865)
Lithoglyphus Buschii Frauenfeld, 1865, ex
Dunker, in litt.: 530, pl. 11
Potamolithus buschii, Pilsbry & Rush 1896:
80
Potamolithus buschii, Pilsbry, 1896: 88
Potamolithus buschii, Pilsbry, 1911: 580, pl.
40, figs. 11-14, pl. 41b, fig. 2
Potamolithus buschii, Parodiz, 1965: 28, figs.
63-72
Type material: Naturhistorisches Museum,
Vienna, Austria.
Type locality: ‘“Erst kürzlich von Buenos-Ay-
res [sic. Colonia Department, Uruguay] er-
halten. Wird gefunden an der Mündung des
St. Juan in den La Plata.”
Description
The shell is imperforate, solid, subglobose
to globose in shape (Figs. 8-10). The shell is
green, with irregular buff zigzag streaks (Fig.
11); some specimens (27% at Anchorena
beach) have a dusky-brown band located su-
tural and peripheral on the body whorl (Fig.
11). The surface is smooth, although marked
with growth-lines (Fig. 8F). The spire length is
variable, between 9.60% and 15% of the
shell length. The number of whorls is most
frequently between 3.75 and 4.00 (Table 4),
convex (penultimate whorl convexity = 0.17
and body whorl convexity = 0.21) inoutline.
The body whorl can be carinate,strongly an-
gular, with a rounded angle, or globose at the
basal periphery (Figs. 8A-D, 10). The body
whorl is convex above the basal periphery,
usually having a low keel or rounded angle at
the back and a short distance below the su-
ture (Fig. 9A-C). There is also, sometimes, a
second spiral ridge below the upper one and
a concavity between both called sulcus (Fig.
9D). The base is flattened in dorsal view (Fig.
9A-C). The aperture is oblique, inclined about
8 LÓPEZ ARMENGOL
P agapetus
М= 20
Number of individuals
19 20 21 22° 23 24 25 26
27 28 29
Number of gill filaments
P buschii
N=18
30, 31 32 33 3403536
FIG. 6. Gill filament number in P. agapetus and Р. buschii. Scatter-diagram between number of gill filaments
and number of individuals.
40° to 54° (X = 46°53’) towards the axis of
coiling; basally rounded and angular at the
top. Columella narrow and flattened or con-
vex (Fig. 8E).
In shells with a discontinous peristome, the
outer lip is thin and may or may not have an
umbilical area. When the umbilical area is
present, it is narrow and bounded by an an-
gle. In shells with a continous peristome, the
inner lip is heavily calloused and the outer lip
is thick (Fig. 8E). Sometimes the peristome is
edged with a black line. There is a well-de-
veloped concave umbilical area bounded by
an angle or an acute ridge. Some specimens
have an umbilical opening.
Statistics on shell dimensions for males
and females of 3.50 to 3.75 whorls are given
in Table 5. No sexual dimorphism in shell size
was evident; females and males at the same
number of whorls have the same size (Fig.
3B).
There was no significant difference in the
number of males and females at Anchorena
Beach (0.90:1).
The entire head is black (melanin), and
there is a black band in the middle of each
tentacle. Next to the eyes there is a hyaline
band with white spheric granules on it (Fig.
12А).
Тре nuchal node is located to the right of
the mid-line and is 0.06 mm high (Fig. 13A,
B). The distance between the base of nuchal
node and the lobes of the eyes is X = 1.01
mm + 0.24 (0.63-1.26). The angle of the base
of nuchal node (with respect to the mid-line
of the neck) is x = 48° (27°-69°).
The penis is simple, without appendages
(Fig. 13C). The penis bears two parallel
bands of melanin running on both sides, one
dorsal along the distal part and the other ven-
tral (Fig. 12B). The distance between the
base of penis and the lobes of the eyes is X =
0.59 mm +0.08 (0.45-0.75). The angle of the
base of the penis (with respect to the mid-line
of the neck) is X = 32° (20-457).
There are 28 to 36 gill filaments (Fig. 6),
with no indication of sexual dimorphism in
their number (X = 30.14 + 1.57 and 32.45 +
1.69 for males and females respectively).
Radula tipically taenioglossate (Fig. 14).
The statistics and cusps formulae given in
Table 3. Distinctive features are: that the mid-
dle of the anterior cusps of the central tooth
is flat; the external edge of lateral angle is
TWO RELATED SPECIES OF POTAMOLITHUS 9
CPE
FIG. 7. Radula of P. agapetus. A, Section of radular ribbon excluding left outer marginals. B, enlargement
of central and right lateral teeth. C, central tooth. D, central and left lateral teeth. E, left lateral and marginal
teeth. F, right inner and outer marginal teeth. Scale bar A = 50 um; B-F = 10 um.
TABLE 3. Formulae for the most common cusps arrangements for the four radular teeth
of P. agapetus and P. buschii.
Tooth N Formula (%)
P. agapetus (4 radulae)
Central 29 6-1-6 6-1-5 5=1-6
(79.30), (17.24), (3.45)
3-3 3-3 3—3
Lateral 38 4—1—5 (44.74); 5- 1-4 (31.58); 5- 1-5 (23.68)
Inner marginal 35 1822
Outer marginal 21 17—23
Р. buschii (2 radulae)
Central 51 4-1-4 4-1-4 4-1-5
(33.30); (33.30); (33.30)
2—2 2=3 222
Lateral 46 3—1-3 (80.43); 4-1—3 (10.87); 2-1-2 (8.70)
Inner marginal 31 9—11
Outer marginal 24 1215
10 LÓPEZ ARMENGOL
FIG. 8. Scanning electron micrographs of the shell of P. buschii. A-D, frontal view. Body whorl: A, carinated;
B, strongly angular; C, rounded angle; D, globose. E, umbilical view. F, enlargement of the shell showing
the surface marked with growth-lines and some pits. Scale bar A-E = 1 mm, F=50' um:
TWO RELATED SPECIES OF POTAMOLITHUS 11
FIG. 9. Scanning electron micrographs of the shell of P. buschii. A-C, dorsal view. Note the flat body whorl
base and the different degrees of subsutural carination: A, carinated; B, angular; C, globose. D, lateral view:
note the sulcus between two ridges. Scale bar = 1 mm.
straight, and the innermost pair of basal
cusps arise from the face of the tooth (Fig.
14C). The ventral part of basal cusps is a little
concave and the basal process is not prom-
inent. The central blade of lateral teeth wid-
ened with respect to the other cusps (Fig.
14A, B, D).
DISCUSSION AND CONCLUSIONS
Potamolithus agapetus shows marked
secondary sexual dimorphism in shell shape
and size. The shape of the body whorl in
males is usually rounded, whereas the female
is subglobose, with a rounded angle at short
distance below the suture and another angle
at the basal periphery. Like other gastropods
showing sexual dimorphism, the female shell
is larger than the male shell.
Potamolithus agapetus was described by
Pilsbry (1911) as the smallest Potamolithus
known, with body whorl evenly rounded,
without keels or angles but his description
did not include subglobose shells. Two
LÓPEZ ARMENGOL
232883
BABA
FIG. 10. Variation in the shape of the shell in P. buschii. Scale bar = 1 mm.
rounded angles are usually present in fe-
males.
Potamolithus buschii was described by
Frauenfeld (1865) as having a wide, carinate
shell, but in subsequent descriptions by Pil-
4mm
FIG. 11. Shell of P. buschii, showing the peripheral
band and irregular buff zigzag streaks.
bry (1911) and Parodiz (1965) the concept of
this species changed. Pilsbry (1911) included
the least angular forms of P. buschii from Isla
San Gabriel (type locality of P. agapetus) and
Parodiz (1965) stated that carinated shells
were not the common form of the species.
Because P. agapetus and P. buschii are
related species and sympatric in Rio de la
Plata, it is probable that P. agapetus females
have been included in the descriptions of P.
buschii by Pilsbry (1911) and Parodiz (1965).
For example, Pilsbry (1911) showed in his
Plate 40, fig. 14, the least angular form of P.
buschii, which is very similar to the female
form of P. agapetus. This is became both
species share the body whorl shape ranging
from angulose to globular, and broad um-
bilical area circled by an angular or acute
ridge. The features that reliably to distinguish
both species, as redefined herein are listed in
Table 6.
ACKNOWLEDGEMENTS
| wish to express my gratitude to Analia
Amor, Instituto de Embriologia, Biologia e
TWO RELATED SPECIES OF POTAMOLITHUS
13
TABLE 4. Whorl number of P. buschii. Frequency of males and females at each whorl stage present at
the localities studied. % = percentage of population.
Whorls
eroded
2.25
3.00
3:25
3.50
3.75
4.00
4.25
N =
Anchorena Beach
3 2 % 3
25 19 15.23 =
1 3 1.38 1
14 el 6.23 ==
64 56 41.52 9
29 68 33.56 1
2 4 2.08 5
135 154 16
Rio San Juan
Isla San Gabriel
3 2
20 19
= 3
3 8
15 24
11 19
32 53
21 37
1 4
103 167
TABLE 5. Shell measurements (mm) and ratios for 78 males and 53
females of 3.50 to 3.75 whorls of P. buschii. X + standard deviation
(range). There is no significant difference between sexes, P < .001.
Characters
Shell length
Body whorl length
Spire length
Shell width
Aperture length
Aperture width
Columella width
Umbilical area width
Shell length/body whorl length
Body whorl length/shell width
Shell length/shell width
Aperture length/shell length
Histología, and Miguel O. Manceñido, Facul-
tad de Ciencias Naturales y Museo, for their
valuable help and criticism of the manuscript.
| am indebted to G. M. Davis and an anony-
males
3.90 + 0.39
(2.84 — 4.5)
3.49 + 0.35
(2.52 — 4.14)
0.41 + 0.09
(0.18 — 0.63)
3.92 + 0.39
(2.52 — 4.68)
3.03 + 0.29
(2.08 — 3.51)
2.22 + 0.23
(1.39 — 2.61)
0.32 + 0.08
(0.09 — 0.45)
0.24 + 0.12
(0.04 — 0.54)
1.11 + 0.02
(1.06 — 1.20)
0.89 + 0.04
(0.79 — 1.03)
1.01 + 0.05
(0.89 — 1.13)
0.78 + 0.05
(0.67 — 0.92)
females
3.77 + 0.45
(2.34 — 4.68)
3.39 + 0.41
(2.16 — 4.32)
0.39 + 0.08
(0.18 — 0.63)
3.87 + 0.48
(2.61 — 4.86)
2.99 + 0.31
(2.16 — 3.69)
2.17 + 0.26
(1.35 — 2.70)
0.34 + 0.08
(0.18 — 0.54)
0.25+0.13
(0.09 — 0.54)
1.11+#0.02
(1.06 — 1.16)
0.88 + 0.03
(0.82 — 0.97)
1.02 + 0.04
(0.94 — 1.11)
0.79 + 0.04
(0.69 — 0.92)
mous referee for critically reading the тапи-
script. | also want to thank Maria |. Braca-
monte (CONICET) for the preparation of rad-
ular material.
14
LÓPEZ ARMENGOL
FIG. 12. Pigment patterns of P. buschii. A, head-neck, dorsal view. B, penis, right side. Scale bar = 1 mm.
TABLE 6. Characters distinguishing P. agapetus and P. buschii.
Characters
Shell
Irregular buff zigzag streaks
Growth-lines
Body whorl sculpture
Sulcus in dorsal view
Body whorl base in dorsal view
Relationship between shell length
and shell width
Aperture inclination
Columella
Peristome
External Features
Head pigment pattern
Eyebrows position
Nuchal node size
Penis pigment pattern
Gill Filaments
Gill filaments number range
Radula
Central teeth
Middle of the anterior cusps
External edge of lateral angle
Ventral part of basal cusps
Basal process prominent
Lateral teeth
Central blade more developed
Sexual Dimorphism in Shell
P. agapetus P. buschii
no yes
faintly marked marked
rounded basal angle
in females
no
concave
longer than wider
35° to 42°
wide
simple and thin
unpigmented
above and around the eyes
0.25 mm
black spot in distal end
19-28
concave hollow
sometimes curved
concave
yes
no
yes
rounded angle, strongly angular,
or carena subsutural and basal
in both sexes
no/yes
flat
wider than longer
40° to 54°
narrow
thicker, sometimes dark-edged
entirely black
in hyaline bands
0.06 mm
two parallel bands
28-36
flat
straight
less concave
no
yes
no
TWO RELATED SPECIES OF POTAMOLITHUS
FIG. 13. Scanning electron micrographs of the
head-neck of P. buschii. A, dorsal view of female
head-neck, showing the nuchal node (arrow). B,
right side of the female head-neck. C, left side of
the head-neck and fully erect penis of a male.
Scale bar A, B = 200 um; C = 500 um.
15
LÓPEZ ARMENGOL
FIG. 14. Radula of P. buschii. A, section of radular ribbon excluding left outer marginals. B, enlargement of
central and lateral teeth. C, central teeth. D, lateral teeth. E, left inner and outer marginal teeth. F, right inner
and outer marginal teeth. Scale bar A = 50 um; B-F = 10 um.
LITERATURE CITED
DAVIS, G. M. & M. C. PONS DA SILVA, 1984, Pot-
amolithus: morphology, convergence, and rela-
tionships among hydrobioid snails. Malacologia,
25: 73-108.
DAVIS, G. M., N. V. SUBBA RAO 4 K. E. HOAG-
LAND, 1986, In search of Tricula (Gastropoda:
Prosobranchia): Tricula defined, and a new ge-
nus described. Proceedings of the Academy
of Natural Sciences of Philadelphia, 138: 426-
442.
EMBERTON, K. C., 1985, Seasonal changes in the
reproductive gross anatomy of the land snail Tri-
odopsis tridentata tridentata (Pulmonata: Po-
lygyridae). Malacologia, 26: 225-239.
FRAUENFELD, G. R. VON, 1865, Zoologische Mis-
cellen. V. Verhandlungen der K. K. Zoologisch-
Botanischen Gesellschaft in Wien, 15: 525-536.
HERSHLER, R. & J. J. LANDYE, 1988, Arizona Hy-
drobiidae (Prosobranchia: Rissoacea). Smithso-
nian Contributions to Zoology, 459: 63 pp.
ICZN, 1994, Opinion 1779. Potamolithus Pilsbry
and Rush, 1896 (Mollusca, Gastropoda): placed
TWO RELATED SPECIES OF POTAMOLITHUS We
on the Official List with Paludina lapidum d’Or-
bigny, 1835 as the type species. Bulletin of Zoo-
logical Nomenclature, 51: 271-272.
KABAT, A. R., 1993, Comments on the proposed
designation of Potamolithus rushii Pilsbry, 1896
as the type species of Potamolithus Pilsbry,
1896 (Mollusca, Gastropoda) (1). Bulletin of Zoo-
logical Nomenclature, 50: 52.
KABAT, A. R. & R. HERSHLER, 1993, The proso-
branch snail family Hydrobiidae (Gastropoda:
Rissooidea): review of classification and su-
praspecific taxa. Smithsonian Contributions to
Zoology, 547: 94 pp.
LOPEZ ARMENGOL, M. F., 1985, Estudio
sistemático y bioecolögico del género Potamo-
lithus (Hydrobiidae) utilizando técnicas numéri-
cas. Facultad de Ciencias Naturales y Museo,
UNLP, Tesis No. 455: 281 pp. unpublished.
LOPEZ ARMENGOL, M. F. & M. O. MANCENIDO,
1992, Potamolithus Pilsbry, 1896 (Mollusca,
Gastropoda): proposed confirmation of P. rushii
Pilsbry, 1986 as the type species. Bulletin of
Zoological Nomenclature, Case 2801, 49: 109-
MS
MANCENDO, М. O. 8 M. Е. LÓPEZ ARMENGOL,
1993, Comments on the proposed designation
of Potamolithus ruhii Pilsbry, 1896 as the type
species of Potamolithus Pilsbry, 1896 (Mollusca,
Gastropoda) (2). Bulletin of Zoological Nomen-
clature, 50: 53.
PARODIZ, J. J., 1965, The hydrobid snails of the
genus Potamolithus (Mesogastropoda-Rissoa-
cea). Sterkiana, 20: 1-38.
PILSBRY, H. A., 1896, Notes on new species of
Ammicolidae collected by Dr. Rush in Uruguay.
The Nautilus, 10: 86-89.
PILSBRY, H. A., 1911, Non-marine Mollusca of Pa-
tagonia. In: W.B. SCOTT, ed., Reports of the
Princeton University Expeditions to Patagonia,
1896-1899, 3(2)(5): 566-602.
PILSBRY, H. A. & W. H. RUSH, 1896, List, with
notes, of land and fresh water shells collected by
Dr. Wm. H. Rush in Uruguay and Argentina. The
Nautilus, 10: 76-81.
Revised Ms. accepted 1 January 1995
MALACOLOGIA, 1996, 38(1-2): 19-31
RECRUITMENT OF DREISSENA POLYMORPHA: DOES THE PRESENCE
AND DENSITY OF CONSPECIFICS DETERMINE THE RECRUITMENT DENSITY
AND PATTERN IN A POPULATION?
М. E. Chase & В. С. Bailey
Ecology and Evolution Group, Department of Zoology, University of Western Ontario,
London, Ontario, Canada N6A 5B7
ABSTRACT
Results of a field experiment conducted to examine the density and spatial pattern of re-
cruitment in a population of Dreissena polymorpha in Lake St. Clair were consistent with the
hypothesis that recruitment is in response to a chemical cue released by conspecific adults.
The number of recruits were significantly higher in treatments in which conspecific adults were
present. Analysis of the distribution of adults and recruits in low and high density treatments
showed a strong spatial correlation between adults and recruits. However, the distribution of
recruits in the low density treatment was more aggregated in comparison to the high density
treatment. Comparison of density and size distribution of recruits between low and high density
treatments and the adjacent natural population revealed recruits in the natural population were
smaller and less dense than recruits in the experimental treatments. This results suggests that
although recruitment is in response to conspecific adults, recruitment into a population with
lower adult densities, as represented by the experimental treatments, may result in enhanced
growth of the new recruits.
Key words: Dreissena polymorpha, recruitment, spatial, density, conspecific.
INTRODUCTION
Larval settlement and juvenile recruitment
are the initial processes determining the
structure of populations of many sessile,
aquatic species (Rodriguez et al., 1993). Suc-
cessful recruitment of a sessile organism de-
pends on the behavioral adaptation of early
life stages to meet or avoid biological and
physical hazards (Schubart et al., 1995). The
location of settlement and potential recruit-
ment can affect the performance and ultimate
survival of a sessile organism. Stimuli neces-
sary for settlement involve a combination of
factors, including speed of fluids and con-
tours of the substratum (e.g., Sebens, 1983;
Wethey, 1986; Butman, 1989; Pawlik & Had-
field, 1990; Pawlik et al., 1991; Johnson,
1994), luminosity (e.g., Crisp & Ritz, 1973;
Young 4 Chia, 1982) and chemical cues (e.g.,
Morse 8 Morse, 1984, Pawlik, 1986; Rai-
mondi, 1988). Perhaps the most widely ex-
amined of all settlement stimuli are the exis-
tence of chemical inducers associated with
conspecific adults. Such cues are of great
ecological importance, because the induction
of settlement by conspecifics can account for
the aggregated distribution of many benthic
marine invertebrates (Rodriguez et al., 1993).
19
Aggregated distributions may increase the
probability of fertilization in individuals that
either release their gametes into the water
column (e.g., Pearse & Arch, 1969; Russo,
1979; Pawlik, 1986) or have internal fertiliza-
tion (e.g., Raimondi, 1991). Aggregation also
acts as an effective defense mechanism (e.g.,
Garnick, 1978; Bernstein et al., 1981; Pawlik,
1986; Hoffman, 1989), increases filter-feeding
efficiency (e.g., Barnes 4 Powell, 1950) and
results in decreased juvenile mortality (e.g.,
Highsmith, 1982). Settlement induced by con-
specific adults has been described in several
benthic invertebrates, including polychaetes
(Jensen & Morse, 1984; Pawlik, 1986), bar-
nacles (Knight-Jones, 1953; Rittschof et al.,
1984; Raimondi, 1988; Johnson 4 Strath-
mann, 1989; Crisp, 1990; Raimondi, 1991),
echinoids (Highsmith, 1982; Burke, 1984) and
molluscs (Seki & Kan-no, 1981).
The majority of studies of settlement and/
or recruitment, however, have been confined
to marine invertebrates. This is a reflection of
the common planktonic larval stage charac-
teristic of many benthic marine invertebrates.
The recent invader Dreissena polymorpha
(Pallas), the zebra mussel, is one of the few
North American freshwater benthic inverte-
brates with a planktonic larval stage. Other
20 CHASE & BAILEY
freshwater bivalves that possess a plank-
tonic larval or juvenile stage include such ex-
otic species as the quagga mussel, Dreis-
sena bugensis, and the asian clam Corbicula
fluminea. Most species of North American
freshwater bivalves reproduce either via a
specialized parasitic larval stage called
glochidia (e.g., Unionidae) or through incuba-
tion of a small number of embryos that simply
crawl away once ready for juvenile existence
(e.g., Sphaeriidae) (Mackie, 1991). Dreissena
polymorpha larvae may remain in the water
column for 5 days to 5 weeks (Sprung, 1993)
before settling onto hard substrata, undergo-
ing metamorphosis and becoming juveniles.
The incorporation of a new cohort or age
class into the population is the stage of the
larval life cycle referred to as recruitment
(Connell, 1985).
Since its introduction into North America,
researchers have devoted considerable en-
ergy to studying the ecology and the control
of D. polymorpha. Extensive research has
been conducted to determine the distribution
(e.g., Hebert et al., 1991; Schaner et al.,
1991; Dermott 8 Munawar, 1993), predict the
spread (e.g., Strayer, 1991; Neary & Leach,
1992: Ramcharan et al., 1992) and ultimate
impact (e.g., Maclsaac et al., 1992; Bunt et
al., 1993) of D. polymorpha on lake ecosys-
tems. However, in order to predict the spread
or impact of D. polymorpha, we must first
understand what factors regulate popula-
tions. For a sessile organism, population dis-
tribution is determined by dispersal ability
and the extent of passive transport at a large
spatial scale and suitable settlement sites at
a small spatial scale (Minchinton & Scheib-
ing, 1991). As a result, a more appropriate
predictor of population structure and com-
munity patterns may be sought through the
study of recruitment.
The results of a 1989 survey of D. polymor-
pha in Lake St. Clair (Hebert et al., 1991) re-
vealed a marked heterogeneity in size and
cohort structure among sites, depending on
the density of D. polymorpha. Hebert et al.
(1991) suggested that veliger settlement may
be cued by a chemical released by conspe-
cific individuals that is an attractant at low
concentrations and a repellent at high con-
centrations. Wainman et al. (1995) also sug-
gested that shell induced factor was the ex-
planation for the difference in recruitment
between experimental substrata with and
without mussels present. Their experimental
design, however, consisted of racks sus-
pended below the water surface in the metal
forbay of a thermal generating station, and
therefore their results may not be represen-
tative of recruitment in the natural population.
The objectives of this study were twofold.
Firstly, we determined whether or not the
presence of conspecifics may be a cue to
induce recruitment into a population of D.
polymorpha at Lake St. Clair. Secondly, we
determined whether or not the spatial ar-
rangement of recruits was influenced by the
presence and density of conspecifics. Re-
sults of recruitment in the experimental study
were chen compared to recruitment in the
natural population in Lake St. Clair.
METHODS
Study Site
The study site was approximately 1 km
from shore at 42°19’57.0’N, 82°33’19.5’W at
2.5 m depth near Stoney Point, Ontario, on
the southeastern shore of Lake St. Clair
(Fig. 1). Lake St. Clair is the smallest of the
Great Lakes, with a total area of 1,114 km?
(Bolsenga & Herdendorf, 1993). The mean
depth is only 3 m, with maximum natural
depth of 6.4 m and maximum depth along a
dredged shipping channel of 8 m (Bolsenga &
Herdendorf, 1993). Average annual tempera-
ture is 11.9°C. Temperatures range from near
freezing for most of the winter to their sum-
mer average peak of 24°C in July and Au-
gust. Substratum at the site was predomi-
nately silt and clay, with some fine sand and
approximately 40% hard substrata, consist-
ing of mainly rocks and Unionidae shells.
Dreissena polymorpha were found on most
submerged hard substrata. Densities of D.
polymorpha at this site (+ SE) were 15,735 (+
316)/m? (1992), 15,545 (+ 310)/т? (1993) and
10,264 (+ 109)/m* (1994) (Chase, unpub-
lished data). Recruitment occurred in August
and October in 1992, August in 1993, and in
October in 1994. Population data from previ-
ous years showed good recruitment at this
site (1992: 5,469 individuals/m?; 1993:
10,393 individuals/m* (Chase, unpublished
data).
Experimental Design
The experimental substrata consisted of
plexiglass plates (18 x 18 cm). Each plate
was attached to a cement block with a stain-
RECRUITMENT OF DREISSENA POLYMORPHA 21
MICHIGAN
LAKE
ST. CLAIR
WINDSOR
ONTARIO
LAKE ERIE
STONEY POINT
FIG. 1. Location of study site at Stoney Point, Ontario, Canada.
less steel bolt (Fig. 2b). The plates were im-
mersed in lake water 2-3 days before use to
remove any manufacturing chemicals that
might have been present and which could
prevent initial attachment by adult mussels.
On May 15, 1994, adult mussels (6-21 mm
in length) were collected from Lake St. Clair
using SCUBA, and subsequently returned to
the laboratory. Mussels were randomly cho-
sen and placed on the plexiglass plates in an
aquaria to allow byssal thread attachment.
Three experimental treatments were estab-
lished:
(1) No adult mussels,
(2) Low adult density (average 167 + 30 in-
dividuals/m?), and
(3) High adult density (average 4583 + 419
individuals/m?).
Mussels on the plates remained in the lab-
oratory for 48 hours, during which time they
were fed dried Chlorella sp. (Beta Green, Na-
trol) ad lib. It was found that moderate tem-
perature (approximately 15°C) and food ad-
dition enhanced byssal thread attachment of
the adults (M. Chase, personal observation).
Data Collection
On May 17, 1994, the plates were ran-
domly arranged in Lake St. Clair using
SCUBA in a 6 x 3 configuration, with a 1-m
perpendicular distance between blocks (Fig.
22 CHASE & BAILEY
im
7 zu 2
(=) эй
no adult mussels high density treatment № low density treatment
plexiglass plate
stainless steel bolt
Be
adult mussel
cement block
6 ст
FIG. 2a. Schematic diagram of experimental design, which consisted of three treatments of adult mussel
density—no adults, low density and high density—in Lake St. Clair. FIG. 2b. Diagram of one cement block
as placed in the field with a plexiglass plate (18 x 18 cm) attached with a stainless steel bolt. FIG. 2c.
Diagram of a plexiglass plate from the low density treatment showing division into 324 (1 x 1 cm) squares
and subsequent analysis at three spatial scales; 1 x 1 cm, 2 x 2 ст and 6 x 6 cm. Open symbols represent
adult mussels, closed symbols represent recruits.
RECRUITMENT OF DREISSENA POLYMORPHA 23
2a). The plates were monitored visually twice
monthly for recruitment.
The plates were removed on November 16,
1994, 182 days after deployment, and re-
turned to the laboratory for examination. Of
the 18 plates deployed, 16 were recovered,
including 6 plates from the high density treat-
ment, 5 plates of the low density treatment,
and 5 plates of the no mussel treatment. In
addition to retrieving the plates, 10 rocks
were randomly collected from the surround-
ing area so that the density and size distribu-
tion of recruits from the experimental treat-
ments could be compared to the natural
population.
In the laboratory, each plate was divided
into 324 (1 x 1 cm) squares (Fig. 2c). The
number of adults and recruits in each square
was recorded under 10x magnification using
a Wild-Heerbrug microscope. Recruitment
was defined as individuals between 0.8 and 4
mm in shell length. Adults and recruits were
then removed from the plates.
Mussels from the natural population were
removed from each of the rocks collected and
preserved in ethanol. Densities of adults and
recruits in the natural population were deter-
mined using the method of Bailey et al. (1995).
The shell length of the recruits from both the
plates and the natural population were mea-
sured at 6.4x magnification using a digitizing
tablet interfaced with an IBM personal com-
puter (Roff & Hopcroft, 1986). Length was
measured as the longest distance between
the umbo and the ventral margin.
Data Analysis
Counts of the number of recruits per plate
in each treatment were Log,, (x + 1) trans-
formed, and a one-way ANOVA was per-
formed followed by a Tukey-Kramer test to
make a posteriori comparisons of means.
Lengths of recruits on the plates were
Log,, transformed and then compared within
and among treatments by use of one-way
ANOVA. Lengths of recruits in each treat-
ment were then compared to Log,, trans-
formed lengths of recruits in the natural pop-
ulation by one-way ANOVA.
To determine the effect of adult density on
the spatial arrangement of recruits, the dis-
tribution of adults and recruits was examined
at three spatial scales; 1 x 1 cm, 2 x 2 cm and
6 x 6 cm (Fig. 2c). A nested analysis of co-
variance was applied to determine how
adults and recruits covaried at these scales.
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HIGH DENSITY LOW DENSITY NO MUSSELS
TREATMENT
FIG. 3. Number of recruits (Log, (X + 1) trans-
formed) (+ SE) in each of the experimental treat-
ments; no adult mussels (n = 5 plates), low density
(n = 5 plates) and high density (n = 6 plates).
This analysis quantified the strength and na-
ture of the covariation of adults and recruits
on the plates in the low and high density
treatments.
RESULTS
Recruit Density
One-way ANOVA on log,, (x + 1) trans-
formed number of recruits showed significant
(F = 83.82, DF = 2,13, p < 0.001) differences
among the treatments (Fig. 3). Pairwise com-
parisons of mean number of recruits in each
treatment using the Tukey-Kramer test
showed that the number of recruits in the no
adult mussel treatment was significantly
lower than the low density treatment (p <
0.001) and the number of recruits in the low
density treatment was significantly lower
than the high density treatment (p = 0.036)
(Fig. 3). Examination of the relationship be-
tween recruit density and adult density within
treatments revealed a positive linear relation-
ship within the low density treatment (Fig. 4a)
but a negative linear relationship within the
high density treatment. Neither regression
was significant (Low density: DF = 1,3, F =
2.84, г? = 0.49; high density: DF = 1,4, F =
7,814, Г = 0,65),
Size Distribution
One-way ANOVA of length of recruits
between the five plates in the low density
treatment was not significant (F = 1.26,
DF = 4,203, p = 0.286); therefore, lengths
of recruits in the low density treatment were
pooled. Lengths of recruits from the six
№
>
RECRUIT DENSITY ( INDIVIDUALS / m’)
1000 т
Е т— == т
3000 3500 4000 4500 5000 5500 6000 6500
ADULT DENSITY ( INDIVIDUALS / m?)
FIG. 4. Regression of recruit density versus adult
density for the low density (A) and high density (B)
treatments.
plates in the high density treatments were
also pooled, because the one-way ANOVA of
length of recruits between the high density
treatments was not significant (F = 2.12, DF =
5.622. р = 0.062).
One-way ANOVA of length of recruits п the
low and high density treatments was signifi-
cant (F = 4.39, DF = 1.834, р = 0.027). The
mean length of recruits in the low density
treatment (1.89 + 0.03 mm) was significantly
larger than the mean length of recruits in the
high density treatment (1.81 + 0.02 mm), de-
spite the small difference in means.
Because the lengths of recruits in the high
and low density treatments differed, separate
ANOVA’s were performed to compare them
to the natural population. One-way ANOVA
revealed the length of the recruits in both the
low (F = 175.04, DF = 1,353, p< 0.001) and
the high (Е = 197-61, DF = 1,773, р. < 0:001)
density treatments (Fig. 5) were significantly
larger than the mean lengths in the natural
population. The mean length of recruits in the
natural population was 1.33 + 0.03 mm (Fig.
5), about 500 um less than in both experi-
mental treatments.
CHASE & BAILEY
Spatial Arrangement
Across both low and high adult density
treatments, and all plates (Fig. 6), correlation
between adults and recruits was high (r =
0.90 + 0.07). There was no significant differ-
ence between the correlation values at either
the 1 x 1, 2 x 2 or 6 x 6 cm Spatial scales in
the low adult density treatment, although the
mean correlation at the 2 x 2 cm spatial scale
was always the highest (2 x 2: r= 1.14 + 0.08;
6 x 67 т = 0:87 + 0.26). Although и Mais
mathematically impossible in simple correla-
tion analysis, such estimates are possible in
nested covariance analysis. They should just
be interpreted as high correlations at this
scale. One-way ANOVA of correlation at the
different spatial scales in the high adult den-
sity treatment revealed no significant differ-
ence between the 2 x 2 and the 6 x 6 cm
spatial scales (F = 2.04, DF = 1, 10, p = 0.183)
although the mean correlation at the 6 x 6 cm
scale (r = 0.88 + 0.07) was always higher than
the mean correlation in the 2 x 2 cm scale
(r = 0.74 + 0.08). Correlation at the 1 x 1 cm
scale was significantly lower than either the 2
x 2 or the 6 x 6 cm scales in the high density
treatment (Е = 17.01, DF = 2, 15, p< 01001}:
Correlation at the 1 x 1 cm scale was also low
in the low density treatments (mean r = 0.64
+ 0.5). Low correlation between adults and
recruits at the 1 x 1 cm scale reflects the
average length of adult mussels on the
plates, which was 1.03 cm.
DISCUSSION
Recruitment Density
Results of the experimental study showed
that the density of recruitment increased with
the density of adults. Little recruitment was
observed on plates with no adult mussels
present. Several explanations may account
for the pattern of recruitment observed in this
study, including differential deposition and
attachment, post depositional movement,
and differential mortality after settlement
(Johnson, 1994). Because this study exam-
ined only recruitment, it is difficult to deter-
mine which of the possible mechanisms may
be underlying the settlement of D. polymor-
pha at Lake St. Clair. Recruitment is defined
as the arrival of the first cohort or age class
into the population (Connell, 1985), so it in-
cludes any post-settlement movement or
2
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HIGH DENSITY TREATMENT | |]
RECRUITMENT OF DREISSENA POLYMORPHA
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209) density experimental treatments and the adjacent natural
= 631) and low (n =
FIG. 5. Length-frequency histograms of recruit length in the high (n
population (n = 148).
5
1X1
2X2
6X6 1X1
26 CHASE & BAILEY
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E 0.75
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HIGH
LOW
FIG. 6. Correlation (+ SE) between adults and recruits in the high and low density treatments at each of the
three spatial scales; 1 x 1 cm, 2 x 2 cm and 6 x 6 cm.
montality that may have occurred. It is possi-
ble that settlement did occur on the clean
plates but the mussels did not survive to the
time of census. Alternatively, another factor
may have been acting as a deterrent to pre-
vent settlement to the clean plates. Even on
plates with adults, recruitment occurred only
on or very close to the adults. It was ob-
served on several occasions that herbivorous
snails were present on the clean plates. While
the mussels are at no risk of predation from
the snails, their presence may still act as a
deterrent to recruitment. Barnacle recruit-
ment can be reduced in the presence of lim-
pets (Denley 8 Underwood, 1979; Miller,
1986) because of the biological disturbance
(i.e., bulldozing) by the limpets. Johnson 4
Strathmann (1989) demonstrated reduced
settlement of barnacle larvae as a result of
prior occupation of the substratum. Their re-
sults indicated that mucus secretions may
have been responsible for the reduction in
settlement, because they may have affected
the adhesion of the larvae or caused an al-
teration in the existing cues present on the
substratum (Johnson & Strathmann, 1989).
The most likely explanation of the recruitment
pattern observed in this study, however, is
that of differential deposition and attach-
ment, that is, recruitment is in response to a
cue released by conspecific adults. Wainman
et al. (1995) also observed no settlement on
treatments without mussels. In addition to
treatments with and without adult mussels,
Wainman et al. (1995) included treatments
with mussel-sized stones. These treatments
served as a control to ascertain whether re-
cruitment was in response to a chemical cue
released by the adults or simply in response
to the heterogeneity of the substrata. Wain-
man et al. (1995) found settlement and re-
cruitment was significantly lower on mussel-
sized stones than on live mussels. This
pattern was maintained even after 10-12
days despite reduction in the numbers of re-
cruits. This pattern suggests that although
there is post settlement mortality, the pattern
of settlement with conspecifics was main-
tained. A laboratory study on settlement and
metamorphosis of larval zebra and quagga
mussels (Baldwin, 1995) provides further ev-
idence for the presence of a chemical cue
associated with conspecific adults. Baldwin
(1995) found that in the laboratory D. poly-
morpha settled and metamorphosed more
readily on natural substrata (adult shells) and
in water from adult rearing tanks as opposed
to water not exposed to adults. On the basis
RECRUITMENT OF DREISSENA POLYMORPHA 27
of our study and the research described
above, it appears that D. polymorpha re-
sponds to some chemical cue released by
adult conspecifics that enhances settlement
and recruitment. The exact nature of the cue,
however, was beyond the scope of this
study.
Our study also examined the effect of in-
creased density of adult mussels on the den-
sity and spatial pattern of recruitment. Prox-
imity to adults allows for synchronization of
spawning and increased fertilization of
spawned gametes as well as local introduc-
tion of food as a result of adult filtering activ-
ity. Large aggregations have a better chance
of surviving physical disturbance and conse-
quently gain a longer adult life span and over-
all increased fecundity (Pawlik, 1986). How-
ever, at high population densities, organisms
may experience intense intraspecific compe-
tition for space (Wu, 1980; Hui & Moyse,
1987) and resources (Russo, 1979) and in-
creased rates of both predation (Fairweather,
1988) and parasitization (Blower 8 Roughgar-
den, 1989). Therefore, at high population
densities there may be selective pressure for
individuals to avoid conspecifics at settle-
ment (Satchell & Farrell, 1993).
Within this study, recruitment density was
highest in the high density treatment. How-
ever, variation in the adult densities within
each of the high and low density treatments
enabled the examination of the relationship
between recruit density and adult density
within each treatment. This variation is the
result of differential attachment of adult mus-
sels within the laboratory and subsequent
loss of adults during transportation and
placement in the field site. Although the data
are limited, it was observed that within the
high density treatment (n = 6 plates) there is
a negative relationship between adult mussel
density and the density of recruits, whereas
in the low density treatment (n = 5 plates),
there is a positive relationship between adult
mussel density and recruit density. lt is pos-
sible that there is reduced recruitment with
higher adult densities, but the adult densities
employed in the high density treatments
were not large enough to elicit such a re-
sponse. When comparison was made be-
tween the recruitment densities from the ex-
perimental treatments and the natural
population, it was observed that the recruit-
ment density in the natural population was
1,257 + 178 individuals per m? [which is com-
parable to the recruitment density in the low
density treatment (1,198 + 141 individuals per
m?)] but much lower than the recruitment
density in the high density treatment (3,189 +
637 individuals/m*). Adult densities in the
natural population were 8,029 + 506 individ-
uals per m? versus only 4,583 + 419 individ-
uals per m° for the high density treatments.
This suggests that there may be some avoid-
ance of the high adult density in the natural
population.
Spatial Arrangement
Examination of the spatial arrangement by
nested analysis of covariance revealed a
strong correlation between adults and re-
cruits, confirming the observation that at all
scales we tended to find recruits when adults
were present. When the spatial arrangement
was examined on three spatial scales the low
density treatment had the highest correlation
at the 2 x 2 cm scale, whereas the high den-
sity treatment the highest correlation was at
the 6 x 6 cm scale. However, correlation in
the 2 x 2 and the 6 x 6 cm scales were not
significantly different within treatments. This
pattern suggests that while in the low density
treatment the recruits are found closer to the
adults than in the high density treatment,
both treatments show the same conclusion
that the recruitment occurs in response to the
presence of adult conspecifics.
In a patchy environment (represented by
the low density treatment), the recruits must
be close to the adults to obtain whatever
benefit — protection, enhanced feeding —
that such an association would elicit. This is
indicative of the higher correlation at the 2 x
2 cm scale. Hoffman (1989) suggested that
gregarious settlement reduces stress on the
vulnerable meta individual. Clumps of barna-
cles may also influence water flow in a way
that enhances feeding (Barnes & Powell,
1950). However, in a more homogenous en-
vironment (represented by the high density
treatment) such a close association may be
detrimental because of competition for space
and resources and the increased risks of pre-
dation of parasitism. In the high density treat-
ment, the adults and recruits covaried on a
larger scale (6 x 6 cm) than in the low density
treatment, indicating a more uniform distribu-
tion. In addition, the ratio of recruits to adults
was much lower in the high density treatment
(0.8 + 0.2) than the low density treatment (7.5
+ 0.8). Hebert et al. (1991) observed a marked
heterogeneity in size and cohort structure in
28 CHASE & BAILEY
D. polymorpha at different sites in Lake St.
Clair in 1989. Members of the 1988 cohort
had the smallest shell sizes at sites with the
highest density, suggesting that their growth
rates were slowed by intraspecific competi-
tion.
Natural Population
Comparison of the mean length of recruits
revealed that recruits in the natural popula-
tion were significantly smaller than recruits in
either the low or the high density treatments.
The largest mean length of recruits was in the
low density treatment (1.89 mm), which may
suggest that increased competition for food
in the high density environment resulted in
reduced growth of recruits. Such a scenario
will confer an advantage of recruiting into a
low density habitat with either more space to
grow or decreased competition for food with
larger mussels. This observation may also
explain the reduction in growth of mussels at
Lake St. Clair since their introduction. Popu-
lation densities near Stoney Point were only
0.5 and 4,500 individuals per m° in 1988 and
1989 respectively (Hebert et al., 1991). At that
time, Mackie (1991) reported that an overwin-
tering young adult between 1-4 mm in shell
length will attain a shell length of 15 to 20 mm
by the end of the year. Our data have shown
that overwintering young adults of similar
size (1-4 mm) had shell lengths of only 9 mm
(Chase, unpublished data) by the end of the
next year. Population densities at Stoney
Point now exceed 10,000 individuals per m°
(Chase, unpublished data). Similar restriction
in growth rates and survival of recent recruits
of the barnacle Pollicipes polymerus were
determined to be the result of competition
between the established adults and the re-
cruits for food resources (Page, 1986). When
large adults were experimentally removed
from an aggregate, the smaller barnacles
were able to increase rapidly in size (Page,
1986). Larger barnacles may also have inter-
fered with the water flow that brings food to
the smaller barnacles (Page, 1986). In the
mussel Mytilus edulis, Kautsky (1982) also re-
ported that growth was suppressed in small
mussels by increased density of large mus-
sels. However, differences in size distribution
and abundance may also be the result of dif-
ferential recruitment between the experimen-
tal and natural population. This observation
may also be the result of the raised level of
the bricks in the water column, which may
enhance growth of recruits. Pontius & Culver
(1995) found that D. polymorpha higher in the
water column had larger biomass, which may
indicate they were better able to obtain food.
However, the significant difference between
the low and high density treatments suggests
an explanation other than height in the water
column.
Conclusion
It appears that for D. polymorpha at Lake
St. Clair, the presence and density of con-
specifics are important determinants of the
recruitment density and pattern in the popu-
lation. The presence of adult conspecifics
may offer some chemical cue that induces
recruitment into the population. However, re-
cruitment into a low density habitat may be
advantageous because it may enhance the
growth of young recruits. Therefore, in D.
polymorpha there appears to be a tradeoff
between adult densities that are high enough
to provide an attachment site and protection
but low enough to enhance growth and pos-
sibly survival. Larger mussels may have a
better chance at surviving the winter, and be-
cause fecundity is related to size in most
benthic invertebrates (Hughes, 1971; Spight
& Emlen, 1976; Brousseau, 1978; Sprung,
1987; Chase & Thomas, 1995), recruitment
into a low density habitat may also enhance
reproductive output, assuming this size dif-
ferential is maintained.
The objective of this study was to examine
recruitment. As such, the extent of post set-
tlement mortality is unknown. It is possible
the post settlement mortality was higher in
the low density treatments than in either the
high density treatment or the natural popula-
tion. Thus, although our study suggests that
recruitment into low density habitats may be
advantageous because of enhanced growth
and survival, it may have also suffered from a
greater initial mortality. However, in terms of
the ultimate survival and population structure
of D. polymorpha at Lake St. Clair our results
are valid.
ACKNOWLEDGEMENTS
We are grateful to R. Coulas, S. MacPher-
son, J. Mitchell and S. Wolfenden for their
assistance in the field. Special thanks to M.
Topping for reading drafts of this paper and
RECRUITMENT OF DREISSENA POLYMORPHA 29
offering constructive criticism. This research
was funded through Natural Science and En-
gineering Research Council of Canada, On-
tario Ministry of Natural Resources, and the
Great Lakes University Research Fund (Lake
Erie Trophic Transfer) grants to R. C. Bailey.
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BALDWIN, B. S., 1995, Settlement and metamor-
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BLOWER, S. M. & J. ROUGHGARDEN, 1989, Par-
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BOLSENGA, S. J. & C. E. HERDENDORF, 1993,
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RECRUITMENT OF DREISSENA POLYMORPHA 31
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Revised Ms. accepted 28 November 1995
MALACOLOGIA, 1996, 38(1-2): 33-34
ADDITIONAL NOTES ON NOMINA FIRST INTRODUCED BY TETSUAKI KIRA IN
“COLOURED ILLUSTRATIONS OF THE SHELLS OF JAPAN”
Rúdiger Bieler' & Richard E. Petit”
The taxa, both available and unavailable,
first proposed by Tetsuaki Kira in the numer-
ous printings of his ‘‘Coloured Illustrations of
the Shells of Japan” and the English edition,
“Shells of the Western Pacific in Color, Vol. |’
were recently listed by us (Bieler & Petit,
1990). At the time, we discussed 54 species-
group and one genus-group name. Forty
names were found to be available from this
work, although only five of these had been
formally designated as new taxa. The diffi-
culty in recognizing some of these unan-
nounced introductions is demonstrated by
our having to add two new taxa that we over-
looked despite extensive searching and
comparing the various printings and editions
in which some nude names have been intro-
duced. There probably remain still others that
have eluded us. Also, we add additional data
on the previously listed genus-group name.
Laevistrombus Abbott, 1960
Laevistrombus Kira, 1955: 31 (nomen nu-
dum).
Laevistrombus Kira. Abbott, 1960: 47-48
(type species designated: Strombus ca-
narium Linné, 1758).
This name first appeared in the 3rd printing
of the 1st edition of ‘‘Coloured Illustrations of
the Shells of Japan” as a subgenus for two
nominal species of Strombus: S. (L.) canar-
¡um Linné, 1758, and S. (L.) isabella Lamarck,
1822. No description or statement of differ-
entiation was given, as required by ICZN
Code Article 13a, nor was a type species
designated. Subsequent printings remained
unchanged at least through the 6th printing
of the 2nd edition (1963). In the 9th printing of
the 2nd edition (1964), Laevistrombus is ele-
vated to genus-level and L. isabella emended
to L. canarium “forma” isabella. The two in-
termediate printings have not been seen, but
have no effect on this discussion.
When Abbott (1960: 47-48) treated Laevis-
trombus as a subgenus in his monograph
of Strombus, he gave a brief description of
Laevistrombus and designated S. canarium
Linné as its type species. Although Abbott
cited Kira as the author of Laevistrombus, the
name had not previously been available and
must take date and authorship from Abbott,
1960 (ICZN Code Article 50a).
Simplicifusus Kuroda & Habe, 1971
Simplicifusus Kira, 1962: 85 (nomen nudum).
Simplicifusus Kira, 1964: 77 (nomen nudum).
Simplicifusus Kira. Kuroda & Habe, 1971:
282, 184 (type species designated: Fusi-
nus simplex Smith [sic; = Fusus simplex
Е. A. Smith, 1879].
Simplicifusus first appeared in Kira's
“Shells of the Western Pacific in Color”
(1962: 85) as a subgenus of Fusinus for two
species: F. (S.) hyphalus M. Smith and F. (S.)
simplex (Smith) [= Fusinus hyphalus Maxwell
Smith, 1940, and Fusus simplex E. A. Smith,
1879]. We cannot determine exactly when
this name first appeared in the Japanese ver-
sion of this work, ‘‘Coloured Illustrations of
the Shells of Japan, Vol. 1.” It was not in the
6th printing (1963) but was in the 9th printing
(1964). We have not seen the two intermedi-
ate printings. However, Kira (1962, 1964)
gave no description or statement of differen-
tiating characters as required by ICZN Code
Article 13a.
Kuroda & Habe (1971) cited Simplicifus
Kira as a genus (Japanese text, p. 282) and
as a subgenus of Fusinus (English text, p.
184). A description of the genus is given (Jap-
anese text, p. 282), and Fusinus simplex
(Smith) is designated as type species (Japa-
nese p. 282; English p. 184). Because Sim-
plicifusus was not previously an available
name, it must take date and authorship from
Kuroda & Habe, 1971 (ICZN Code Article
50a).
'Field Museum of Natural History, Roosevelt Road at Lake Shore Drive, Chicago, Illinois 60605, U.S.A.
2P. O. Box 30, North Myrtle Beach, South Carolina 29582, U.S.A.
34 BIELER & PETIT
Pictodentalium Habe, 1963
Pictodentalium Kira, 1959: 105 (and subse-
quent years; nomen nudum).
Pictodentalium Habe, 1963: 255 (genus de-
scribed; type species designated: Den-
talium (Pictodentalium) formosum hirasei
Kira, 1959).
In our previous paper (1990: 141), we
showed that this genus-group name was a
nomen nudum in all editions of Kira's works.
lt was treated in a systematic manner by
Habe in 1963 (p. 255), who gave a descrip-
tion of it as a subgenus. He attributed the
name to Kira and gave the type-species as
Dentalium (Pictodentalium) formosum hirasei
Kira, stating that designation was by mono-
typy. He then placed D. (P.) formosum hirasei
Kira in the synonymy of D. (P.) formosum (A.
Adams & Reeve, 1850). Because Pictoden-
talium had not previously been made avail-
able, it must take date and authorship from
Habe, 1963 (ICZN Code Article 50a).
We thank Dr. Alan R. Kabat for bringing to
our attention the omission of Simplicifusus in
our earlier paper, and an anonymous re-
viewer for additional data on the availability
of Pictodentalium.
LITERATURE CITED
ABBOTT, R. T., 1960, The genus Strombus in the
Indo-Pacific. Indo-Pacific Mollusca, 1: 33-146.
BIELER, R. & R. E. PETIT, 1990, On the various
editions of Tetsuaki Kira’s “Coloured Illustra-
tions of the Shells of Japan” and “Shells of the
Western Pacific in Color, Vol. |,” with an anno-
tated list of new names introduced. Malacologia,
32: 131-145.
HABE, T., 1963, A classification of the scaphopod
mollusks found in Japan and its adjacent area.
Bulletin of the National Science Museum (Tokyo),
6: 252-281, pls. 37-38.
[ICZN] International Commission on Zoological No-
menclature, 1985, International Code of Zoolog-
ical Nomenclature, 3rd ed. London, Berkeley,
and Los Angeles, xx + 338 pp.
KIRA, T., 1955, Coloured illustrations of the shells
of Japan. [viii] + 204 pp., 67 pls.; Hoikusha, Os-
aka [for additional printings, see Bieler & Petit,
1990].
KIRA, T., 1959, Coloured illustrations of the shells
of Japan. Enlarged & Revised Edition. [1st print-
ing of Revised Edition: March 10, 1959]. [6] + vii
+ [1] + 239 pp., [1] + 71 pls.; Hoikusha, Osaka.
[6th printing: February 5, 1963; 9th printing: No-
vember 1, 1964; for additional printings, see
Bieler & Petit, 1990].
KIRA, T., 1962, Shells of the western Pacific in
color. [vii] + 224 pp., 72 pls. Hoikusha, Osaka [for
additional printings, see Bieler & Petit, 1990].
KURODA, T. & T. HABE, 1971, [Descriptions of
genera and species] in Kuroda, Habe & Oyama,
The sea shells of Sagami Bay. Maruzen, Tokyo.
xix + 741 pp. [in Japanese], pls. 1-121, 489 pp.
[in English], 51 pp. index, map.
Revised Ms. accepted 28 November 1995
MALACOLOGIA, 1996, 38(1-2): 35-46
ON THE NEW NAMES INTRODUCED IN THE VARIOUS PRINTINGS OF “SHELLS
OF THE WORLD IN COLOUR” [VOL. | BY TADASHIGE HABE AND KIYOSHI ITO;
VOL. Il BY TADASHIGE HABE AND SADAO KOSUGE]
Richard Е. Petit" & Rüdiger Bieler”
ABSTRACT
The two volumes of “Shells of the World in Colour” (Vol. |, “The Northern Pacific” by Habe
& Ito; Vol. Il, “The Tropical Pacific’’ by Habe & Kosuge) contain many gastropod and bivalve
names denoted as new therein. Some of these are nomina nuda made available only in later
publications. However, the volumes also contain new taxa that are made available but not
indicated as such. The problem is compounded by the existence of multiple printings of both
volumes in which unexplained nomenclatural changes have been made. Forty-four species-
group names and two genus-group names date from these works. Twelve genus-group names
indicated as new were not made available until later. All pertinent treatments of these taxa are
listed.
INTRODUCTION
In our continuing efforts to determine the
status and correct dates of publication of
various taxa proposed by Japanese authors,
this paper discusses names introduced in the
two volumes of ‘‘Shells of the World in Co-
lour” (Vol. |, “The Northern Pacific” by Habe
8 Ito; Vol. Il, “The Tropical Pacific” by Habe
& Kosuge). Both of these volumes went
through numerous printings, with changes
being made that are not indicated as such.
Neither book is easy to locate, and few
workers have access to more than one print-
ing (we have failed to locate any copies of
some printings). This paper lists the changes
between printings that affect zoological no-
menclature. At least 14 genus-group and 44
species-group names are involved, spanning
many marine gastropod and bivalve families.
Of particular importance is the determina-
tion of when a particular taxon was made
available for taxonomic purposes. The de-
scriptions of the species and subspecies in
the two works under consideration are in
Japanese and usually very brief. These spe-
cies-group taxa are, however, accepted as
being validly proposed. The genus-group
taxa present more serious problems because
12 of the new names were introduced with-
out fulfilling ICZN Code Article 13 require-
ments of providing a fixation of type species,
and a differentiating description or indication
to such. They are here regarded as nomina
nuda and became available only in later
works. Two names, Harpofusus and Mega-
crenella, appear to fulfill the minimal require-
ments set by the Code and are here ac-
cepted as dating from their first appearance.
It is hoped that the following notes will be
of value to systematists who must refer to
these taxa. We have maintained original or-
thography when possible, and have not indi-
cated some typographical errors and incor-
rect usages in order to avoid using “[sic]” as
much as possible. Readers should be aware
that in addition to these “new” names there
are numerous changes between the editions
involving generic or (for subspecies) specific
allocations, re-identifications, and adjust-
ments in spelling and latinization. The works
apparently were newly typeset, at least in
part, between printings, sometimes resulting
in a compounding of problems. An example
of the combination of intended and acciden-
tal changes is Habe & Ito’s reference to a
species of Neptunea (p. 66, pl. 33, fig. 8); this
was initially identified as Neptunea minor and
later (1977) corrected to “Neptunea Ruro-
sio,’’ a lapsus for N. kuroshio Oyama, 1958.
An example of the taxonomic confusion in
these works is the nominal subspecies shi-
rogai, first introduced as ‘‘Collisella pelta shi-
rogai Habe et Ito (nov.)” in 1965. The 1977
printing of the work, referring to the same
illustration, not only still indicated it as being
'P. O. Box 30, North Myrtle Beach, South Carolina 29582, U.S.A.
“Field Museum of Natural History, Roosevelt Road at Lake Shore Drive, Chicago, Illinois 60605, U.S.A.
36 PETIT & BIELER
new, but changed the name of the species:
“Collisella cassis shirogai Habe et Ito (nov.).”
An additional layer of difficulty was intro-
duced by printer's errors. For instance,
“Buccinum chishimananux Habe et Ito
(nov.)” of 1965a was meant to introduce a
new subspecies, nux, for the species chishi-
manum.
Another example that has perplexed au-
thors is Harpa kawamurai Habe, first intro-
duced in the 3rd printing of Habe & Kosuge
(1972) with no indication that it was new.
Harpa kajiyamai Habe, which appeared at the
same time, has never before been correctly
cited in the literature.
Systematists are urged to cite these works
by printing. The date of a particular printing
can be easily determined from the colophon
(inscription at end of each copy). For details
on date determination, see Bieler & Petit
(1990: 132).
LISTING OF NEW NAMES
(A) Habe 4 Ito, 1965 (in sequence of occur-
rence in volume; the work in which each
taxon is considered to have been made
available is shown by the usage of
1965a, 1965b, or a later date)
1) Collisella pelta shirogai, 1965a
2) Omphalomargarites, 1965b
3) Cirsotrema kagayai, 1965a
4) Bulbus flavus elongatus, 1965a
5) Trophonopsis scitula emphaticus, 1965a
6) Boreotrophon paucicostatus, 1965a
7) Nodulotrophon, 1965b
8) Mohnia multicostata, 1965a
9) Ancistrolepis trochoidea ovoidea, 1965a
0) Fusipagoda, 1965b
1) Buccinum chishimanum nux, 1965a
2) Buccinum hosoyai, 1965a
3) Висстит opisthoplectum microcon-
cha, 1965a
4) Buccinum felis shikamai, 1965a
5) Buccinum kawamurai, 1965a
6) Clinopegma buccinoides, 1965a
7) Neoberingus, 1965b
8) Beringion, 1965b
9) Harpofusus, 1965a
0) Volutopsion, 1965b
1) Buccinum subreticulatum, 1965a
2) Buccinum ferrugineum, 1965a
3) Висстит kinukatsugi Habe 4 Ito, 1968
4) Buccinum midori, 1965a
5) Boreomelon stearnsii ryosukei, 1965a
(26) Fulgoraria (Musashia) kaneko hayashii,
1965a
(27) Decollidrillia, 1965b
(28) Decollidrillia nigra, 1965a
(29) Megacrenella, 1965a
(30) Adula californiensis chosenica, 1965a
(31) Megacardita ferruginosa koreana, 1965a
(B) Habe & Kosuge, 1966 (in sequence of
occurrence in volume; the work in which
each taxon is considered to have been
made available is shown by the usage of
1966a, 1966b, or a later date)
(32) Patelloida (Collisellina) saccharinoides,
1966a
) Astralium yamamurae, 1966a
) Granulittorina, 1966b
) Granulittorina philippiana, 1966a
) Clypeomorus batillariaeformis, 1966a
) Ficadusta, 1966b
) Reticutriton, 1966b
) Spinidrupa, 1966b
) Pyrene testudinaria nigropardalis,
1966a
(41) Pyrene lacteoides, 1966a
(42) Plicarcularia gibbosuloidea, 1966a
(43) Hemifusus carinifer, 1966a
(44) Latirus stenomphalus, 1966a
(45)
(46)
(47)
(33
(34
(35
(36
(37
(38
(39
(40
Vexillum rubrocostatum, 1966a
Nebularia yaekoae, 1966a
Награ kawamurai Habe, in Habe 4
Kosuge, 1972
(48) Harpa kajiyamai Habe, in Habe 4
Kosuge, 1972
(49) Volutoconus grossi mcmichaeli, 1966a
(50) Brachytoma kurodai, 1966a
(51) Brachytoma kawamurai, 1966a
(52) Brachytoma vexillium, 1966a
(53) Eglisia brunnea, 1966a
(54) Mantellum perfragile, 1966a
(55) Anomiostrea, 1966b
(56) Laevicardium rubropictum, 1966a
(57) Vasticardium nigropunctatum, 1966a
(58) Macrotoma yamamurae, 1966a
DISCUSSION BY VOLUME
“Shells of the World in Colour, Vol. |. The
Northern Pacific.” Tadashige Habe and
Kiyoshi Ito
The first printing of ““Shells of the World in
Colour, Vol. |” is dated June 1, 1965 (Habe 4
Ito, 1965a). A paper by Habe & Ito published
in Venus (The Japanese Journal of Malacol-
ogy) on July 31, 1965 (1965b) also contains
SHELLS OF THE WORLD IN COLOR 37
descriptions of taxa, indicated as new
therein, which had been shown as new in the
book. In the next few years there were sev-
eral printings of the book; Dr. Kosuge (per-
sonal comm., March 15, 1995) advises that
the 11th printing appeared in March 1991.
Printings that we have seen:
Printing 1 June 1, 1965 (1965a)
2 September 1, 1970
4 August 1, 1972
5 January 20, 1974
8 October 1, 1977
The following new species appear (using
the original arrangement of families). Impor-
tant changes between printings and refer-
ences from other sources are also listed.
GASTROPODA
Acmaeidae
(1) Collisella cassis shirogai Habe & Ito, 1965a
Collisella pelta shirogai Habe et Ito (nov.).
Habe & Ito, 1965a: 11, pl. 4, fig, 18;
1970, 1972, 1974: ibid.
Collisella pelta shirogai subsp. nov. Habe &
Ito, 1965b: 16, 29, pl. 4, fig. 8.
Collisella pelta shirogai Habe et Ito. Habe,
1977: 111 (cited as of 1965a).
Collisella cassis shirogai Habe et Ito (nov.).
Habe & Ito, 1977: 11, pl. 4, fig. 18.
Trochidae
(2) Omphalomargarites Habe & Ito, 1965b
Omphalomargarites (nov) vorticifera (Dall,
1873). Habe & Ito, 1965a: 17, pl. 6, figs.
6, 7; 1970, 1972, 1974, 1977: ibid. (ge-
nus-group name = nomen nudum).
Omphalomargarites subgen. nov. Habe & Ito,
1965b: 17 (type species Margarites vor-
ticifera (Dall, 1873), with no indication of
genus in which it was to be placed).
Omphalomargarites gen. nov. Habe & Ito,
1965b: 30 (type species, Margarites vor-
ticifera (Dall, 1873).
Omphalomargarites (gen. nov.) vorticifera
(Dall). Habe & Ito, 1965b: 45 (plate cap-
tion for pl. 2).
Omphalomargarites Habe & Ito. Kuroda &
Habe, 1971: 31(21) (with Habe & Ito,
1965b, given precedence over 1965a,
and with type species stated to be by
original designation)
Omphalomargarites Habe et Ito. Habe, 1977:
90 (cited as of 1965a, with type as Mar-
garites vorticifera (Dall, 1873) by mono-
typy; 1965b mentioned only as a “cf.”
reference).
Epitoniidae
(3) Cirsotrema kagayai Habe & Ito, 1965a
Cirsotrema kagayai Habe et Ito (nov.). Habe &
ito, 1965429 pl. 7, fig. 25; 1970, 1972,
1974, 1977: ibid.
Cirsotrema kagayai sp. nov. Habe & Ito,
19656: 17, 30, pl. 2; tig: 9.
Cirsotrema kagayai Habe et Ito. Habe, 1977:
56 (cited as of 1965a).
Naticidae
(4) Bulbus flavus elongatus Habe & Ito, 1965a
Bulbus flavus elongatus Habe et Ito (nov).
Habe & Ito, 1965a: 31, pl. 8, fig. 8; 1970,
1972, 1974, 1977: ibid.
Bulbus flavus elongatus subsp. nov. Habe &
Мо, 19656: 17, 31, pl. 3, fig: 2.
Bulbus flavus elongatus Habe et Ito. Habe,
1977: 38 (cited as of 1965a).
Muricidae
(5) Trophonopsis scitula emphaticus Habe &
Ito, 1965a
Trophonopsis scitula emphaticus Habe et Ito
(nov.). Habe & Ito, 1965a: 36, pl. 10, fig.
10; 1970, 1972, 1974, 1977: ibid.
Trophonopsis scitula emphaticus subsp. nov.
Habe & Ito, 1965b: 18, 31, pl. 2, fig. 1.
Trophonopsis scitulus emphatica Habe et Ito.
Habe, 1977: 38 (cited as of 1965a).
(6) Boreotrophon paucicostatus Habe & Ito,
1965a
Boreotrophon paucicostatus Habe et Ito
(nov: [sic]). Habe 4 Ito, 1965a: 37, pl. 10,
fig. 13; 1970, 1972, 1974, 1977: ibid.
Boreotrophon paucicostatus sp. nov. Habe &
Ito, 1965b: 18, 32, pl. 2, fig. 10.
Boreotrophon paucicostatus Habe et Ito.
Habe, 1977: 95 (cited as of 1965a).
(7) Nodulotrophon Habe & Ito, 1965b
Nodulotrophon (nov.) аа! (Kobelt, 1878).
Habe & Ito, 1965a: 37, pl. 10, fig. 14;
38 PETIT & BIELER
1970, 1972, 1974, 1977: ibid. (genus-
group name = nomen nudum).
Nodulotrophon gen. nov. Habe & Ito, 1965b:
19, 32 (with type species as Trophon
dalli Kobelt, 1878).
Nodulotrophon Habe et Ito. Habe, 1977: 87
(cited as of 1965a, with type, by mono-
typy, Trophon dalli Kobelt, 1878; 1965b
not mentioned).
Taxonomic note: This genus-group name
must date from 1965b because there
was no description in 1965a.
Buccinidae
(8) Mohnia multicostata Habe & Ito, 1965a
Mohnia multicostata Habe et Ito (nov.). Habe
& Ito, 1965а: 45, pl. 13, fig. 12; 1970,
1972, 1974, 1977: ibid.
Mohnia multicostata sp. nov. Habe & Ito,
1965b: 19, 33, pl. 2, fig: 2.
Mohnia multicostata Habe et Ito. Habe, 1977:
80 (cited as of 1965a).
(9) Ancistrolepis trochoidea ovoidea Habe &
Ito, 1965a
Ancistrolepis trochoideus ovoideus Habe et
Ito (nov.). Habe & Ito, 1965a: 46, pl. 13,
fig. 18; 1970, 1972, 1974, 1977: ibid.
Ancistrolepis trochoideus ovoideus subsp.
nov. Habe & Ito, 1965b: 20, 33, pl. 2, fig.
13%
Ancistrolepis trochoidea [Bathyancistrolepis]
ovoidea Habe et Ito. Habe, 1977: 92
(cited as of 1965a).
(10) Fusipagoda Habe 4 Ito, 1965b
Fusipagoda (nov.) exquisita Dall, 1913. Habe
& Ito, 1965a: 48; Habe & Ito, 1970, 1972,
1974, 1977: ibid. (genus-group name =
nomen nudum).
Fusipagoda gen. nov., Habe & Ito, 1965b: 21
(with type species as Mohnia exquisita
Dall).
Fusipagoda Habe et Ito. Habe, 1977: 43
(cited as of 1965b with type species as
cited, by original designation; 1965a
cited as “name only’’).
(11) Buccinum chishimanum nux Habe 4 Ito,
1965a
Buccinum chishimananux [sic] Habe et Ito
(nov.). Habe 4 Ito, 1965a: 49, pl. 14, fig.
2.
Buccinum chishimanum nux subsp. nov.
Habe & Ito, 1965b: 22, 36, pl. 2, fig. 7.
Buccimum [sic] chishimana nux Habe et Ito
(nov.). Habe 4 Ito, 1970: 49, pl. 14, fig. 1;
1972, 1974, 1977: ibid.
Buccinum chishimanum nux Habe et Ito.
Habe, 1977: 88 (cited as of 1965).
(12) Buccinum hosoyai Habe & Ito, 1965a
Buccinum hosoyai Habe et Ito (nov.). Habe &
Ito, 1965a: 49, pl. 14, fig. 2; Habe & Ito,
1970, 1972, 1974, 1977: ibid.
Buccinum hosoyai sp. nov. Habe & Ito,
1965b::23,:36, pl. 2599-89:
Buccinum hosoyai Habe et Ito. Habe, 1977:
49 (cited as of 1965a).
(13) Buccinum opisthoplectum microconcha
Habe & Ito, 1965a
Buccinum opisthoplectum microconcha
Habe et Ito (nov.). Habe & Ito, 1965a: 50,
pl. 14, fig. 7; 1970, 1972, 1974, 197%
ibid.
Buccinum opisthoplectum microconcha
subsp. nov. Habe & Ito, 1965b: 23, 37,
pl. 2, fig. 6.
Buccinum opisthoplectum microconcha
Habe et Ito. Habe, 1977: 75 (cited as of
1965a; stated to be a synonym of Buc-
cinum japonicum A. Adams, 1861).
(14) Висстит felis shikamai Habe 4 Ito,
1965a
Buccinum felis shikamai Habe et Ito (nov.).
Habe 4 Ito, 1965a: 50; 1970, 1972, 1974,
1977: ibid.
Buccinum felis shikamai subsp. nov. Habe &
Ко, 19655: 23; 37, pl. 2, fig 5.
Buccinum felis shikamai Habe et Ito. Habe,
1977: 110 (cited as of 1965b).
(15) Buccinum kawamurai Habe 4 Ito, 1965a
Buccinum kawamurai Habe et Ito (nov.).
Habe & Ito, 1965a: 52, pl. 15, fig. 1;
1970, 1972, 1974, 1977: ibid.
Висстит kawamurai sp. nov. Habe & Ito,
1965b: 26, 38, ple 2. fig. it.
Buccinum kawamurai Habe et Ito. Habe,
1977: 58 (cited as of 1965a).
(16) Clinopegma buccinoides Habe & Ito,
1965a
Clinopegma buccinoides Habe et Ito (nov.).
Habe & Ito, 1965a: 55, pl. 16, fig. 1;
1970, 1972, 1974, 1977: ibid.
Clinopegma buccinoides sp. nov., Habe &
Ito, 1965b: 27, 41, pl. 2, fig. 12.
SHELLS OF THE WORLD IN COLOR 39
Clinopegma buccinoides Habe et Ito. Habe,
1977: 28 (cited as of 1965a).
(17) Neoberingius Habe & Ito, 1965b
Neoberingius (nov.) frielei Dall, (1895) [sic].
Habe & Ito, 1965a: 57, pl. 17, fig. 3. (ge-
nus-group name = nomen nudum).
Neoberingius gen. nov. Habe & Ito, 1965b:
21, 35, pl. 3, fig. 7. (type species, Ber-
ingius frielei Dall, 1894 [sic]).
Neoberingius (nov.) frielei (Dall, 1895). Habe
& Ко, 1970: 57, pl 17; fig: 3; 1972, 1974,
1977: ibid.
Neoberingius Habe et Ito. Habe, 1977: 83
(cited as of 1965b, with type species as
cited, by original designation; 1965a
cited as “name only”).
(18) Beringion Habe 4 Ito, 1965b
Beringion (nov.) marshalli (Dall, 1919). Habe &
Ito, 1965a: 58, pl. 17, fig. 4; 1970, 1972,
1974, 1977: ibid. (genus-group name =
nomen nudum).
Beringion gen. nov. Habe & Ito, 1965b: 21,
35, pl. 3, fig. 6 (with type species as Ber-
ingius marshalli Dall, 1919).
Beringion Habe et Ito. Habe, 1977: 27 (cited
as of 1965b, with type species as cited,
by original designation; 1965a referred
to with comments: “‘f. 4 as Beringion
(nov.) marshalli; f. 5, B. beringii, with a
notice of ‘the type-species of Beringion’,
name only’’).
Taxonomic note: Habe’s statement (1977:
83) is ambiguous as the mention of
“type species” in the Japanese text is in
the context of ‘‘Beringion type species is
figured,’ which appears in discussion of
B. beringii (Middendorff). He cited the
new genus as of 1965b, which we con-
sider to be correct.
(19) Harpofusus Habe & Ito, 1965a
Harpofusus (nov.) melonis (Dall, 1891). Habe
& Ito, 1965a: 59, pl. 18, fig. 1; 1970,
1972, 1974: ibid. (with type species, by
monotypy, Harpofusus melonis (Dall,
1891)).
Harpofusus gen. nov. Habe & Ito, 1965b: 20,
34 (with type species as Pyrulofusus
melonis Dall, 1891 [as Pyrofusus on p.
20)).
Pyrulofusus (Harpofusus) melonis (Dall,
1891). Habe & Ito, 1977: 59, pl. 18, fig. 1.
Harpofusus Habe et lto. Habe, 1977: 46.
(listed as a genus of Buccinidae, cited as
of 1965a, with type species, by mono-
typy, Pyrulofusus melonis (Dall, 1891) [=
Strombella melonis Dall, 1891)).
Taxonomic note: Habe £ lto (1965a: 59)
move this species from its previous
placement (Volutopsis, name given in
Japanese only) into a new genus, based
on the yellowish-orange aperture color-
ation and the vertical shell folds and spi-
ral ribs. Similarity to Pyrulofusus is also
mentioned. This fulfills the ICZN Code
requirements, and we date this taxon as
of 1965a.
(20) Volutopsion Habe & Ito, 1965b
Volutopsion (nov.) castaneus (Mórch, 1858).
Habe & Ito, 1965a: 62, pl. 20, fig. 6;
1970, 1972, 1974, 1977: ibid. (genus-
group name = nomen nudum).
Volutopsion gen. nov. Habe 4 Ito, 1965b: 21,
35, pl. 2, fig. 15 (with type species as
Volutopsius castaneus Dall [sic)).
Volutopsion Habe et Ito. Habe, 1977: 131
(cited as of 1965a with type species, by
monotypy, Volutopsion castaneus [-um]
(Mórch, 1858); 1965b is mentioned only
as a “cf.” reference, with same type
species indicated, but by original desig-
nation. We consider this genus name to
be available from 1965b, with type spe-
cies, by original designation, Volutop-
sion castaneum (Mörch, 1858) [= Neptu-
nea castanea Mórch, 1858].
(21) Buccinum subreticulatum Habe & Ito,
1965a
Buccinum Subreticulatum [sic] Habe et Ito
(nov.). Habe & Ito, 1965a: 73, pl. 27, fig.
4.
Buccinum subreticulatum sp. nov. Habe &
Ito, 1965b: 24, 39, pl. 2, fig. 14.
Buccinum subreticulatum Habe et Ito (nov.).
Habe & Ito, 1970: 73, pl. 27, fig. 4; 1972,
1974, 1977: ibid.
Buccinum subreticulatum Habe et Ito. Habe,
1977: 118 (cited as of 1965a).
(22) Buccinum ferrugineum Habe & Ito,
1965a
(23) Buccinum kinukatsugi Habe & Ito, 1968
Buccinum ferrugineum Habe et Ito (nov.).
Habe & Ito, 1965a: 76, pl. 28, fig. 8.
Buccinum ferrugineum sp. nov. Habe & Ito,
1965b: 25, 40, pl. 3, fig. 3.
Buccinum kinukatsugi nom. nov. Habe & Ito,
40 PETIT & BIELER
1968: 2, 5, pl. 1, fig. 4 (new name for
Buccinum ferrugineum Habe & Ito, 1965,
non Born, 1780 [sic; = 1778)).
Buccinum kinukatsugi Habe et Ito (nov.).
Habe & Ito, 1970: 76, pl. 28, fig. 8; 1972,
1974, 1977: ibid.
Buccinum kinukatsugi Habe et Ito. Habe,
1977: 63 (cited as of 1968).
(24) Buccinum midori Habe & Ito, 1965a
Buccinum midori Habe et Ito (nov.). Habe &
Ito, 1965a: 76, pl. 28, fig. 9; 1970, 1972,
1974, 1977: ibid.
Buccinum midori sp. nov. Habe & Ito, 1965b:
25, 405 pl. 2, fig. 16:
Buccinum midori Habe et Ito. Habe, 1977: 75
(cited as of 1965a).
Volutidae
(25) Boreomelon stearnsii ryosukei Habe &
Ito, 1965a
Boreomelon stearnsii ryosukei Habe et Ito
(nov.). Habe & Ito, 1965a: 77, pl. 29, fig.
2; 1970, 1972, 1974: ibid.
Boreomelon stearnsii ryosukei subsp. nov.
Habe & Ito, 1965b: 26, 42, pl. 2, fig. 17.
Boromelon [sic] stearnsii гуозике! Habe et Ito
(nov.). Habe & Ito, 1977: 77, pl. 29, fig. 2.
Boreomelon stearnsii ryosukei Habe et Ito.
Habe, 1977: 103 (cited as of 1965a).
(26) Fulgoraria (Musashia) kaneko hayashii
Habe & Ito, 1965a
Fulgoraria (Musashia) kaneko hayashii Habe
et Ito (nov.). Habe & Ito, 1965a: 77, pl.
29, fig. 4; 1970, 1972, 1974, 1977: ibid.
Fulgoraria (Musashia) kaneko hayashii subsp.
nov. Habe & Ito, 1965b: 26, 42, pl. 3, fig.
oF
Fulgoraria (Musashia) kaneko hayashii Habe
et Ito. Habe, 1977: 47 (cited as of 1965a).
Turridae
(27) Decollidrillia Habe & Ito, 1965b
(28) Decollidrillia nigra Habe & Ito, 1965a
Decollidrillia nigra Hade [sic] et Ito (nov.).
Habe & Ito, 1965a: 80, pl. 30, fig. 6. (ge-
nus-group name = nomen nudum).
Decollidrillia nigra gen. et sp. nov. Habe & Ito,
1965b: 27, 43, pl. 4, fig. 6.
Decollidrillia nigra Habe et Ito (nov.). Habe &
Ito, 1970: 80, pl. 30, fig. 6; 1972, 1974,
1977: ibid.
Decollidrillia Habe et Ito. Habe, 1977: 35
(cited as of 1965b, with type, by original
designation, D. nigra; 1965a cited as
“name only”).
Decollidrillia nigra Hade [-be] et Ito. Habe,
1977: 83 (species name cited as of
1965a).
Taxonomic note: We agree that this new ge-
nus dates from 1965b, but type desig-
nation is by monotypy (Articles 13c,
68d).
Bivalvia
Mytilidae
(29) Megacrenella Habe & Ito, 1965a
Crenella (Megacrenella nov.) columbiana
Dall, 1897. Habe & Ito, 1965a: 109, pl.
35, fig. 11; 1970, 1972, 1974, 1977: ibid.
(with type species, by monotypy,
Crenella (Megacrenella) columbiana
(Dall, 1897)).
Megacrenella gen. nov. Habe & Ito, 1965b:
28, 44, pl. 3, fig. 4 (with type species as
Crenella columbiana Dall, 1897; 1965a
listed as a “cf.” reference).
Megacrenella Habe et Ito. Habe, 1977: 74
(cited as of 1965a, with type species, by
original designation, Crenella columbi-
ana Dall, 1897)
Taxonomic note: We consider the type indi-
cation as by monotypy. The two other
nominal taxa mentioned in the Japanese
text are clearly stated to be synonyms of
Crenella columbiana. Habe & Ito (1965a:
100, in Japanese) refer to something that
translates to “type species group,”
which we cannot accept as original des-
ignation. The authors discuss the posi-
tion of the group, based on morpholog-
ical characters, as standing between
Solamen and Crenella (the latter name
mentioned only in Japanese characters)
and also indicate its relationship to
Arvella. This appears to fulfill the ICZN
Code requirements, and we date this
taxon as of 1965a.
(30) Adula californiensis chosenica Habe &
Ito, 1965a
Adula californiensis chosenica (Kuroda MS.)
Habe et Ito (nov.). Habe & Ito, 1965a:
11а, pl. 327, fig--4= 1970; 197201974
1977: ibid.
SHELLS OF THE WORLD IN COLOR 41
Adula californiensis chosenica subsp. nov.
Habe 4 Ito, 1965b: 28, 43, pl. 3, fig. 1.
Adula californiensis chosenica Habe et Ito.
Habe, 1977: 31 (cited as of 1965a and
stated to be a synonym of A. schmidti
(Schrenck, 1867)).
Carditidae
(31) Megacardita ferruginosa koreana Habe &
Ito, 1965a
Megacardita ferruginosa koreana Habe et Ito
(nov.). Habe & Ito, 1965a: 128, pl. 43, fig.
8; 1970, 1972, 1974, 1977: ibid.
Megacardita ferruginea [sic] koreana subsp.
nov. Habe & Ito, 1965b: 28, 45 (plate
caption), pl. 3, fig. 8.
Megacardita ferruginea [sic] koreanica [sic]
subsp. nov. Habe & Ito, 1965b: 44.
Megacardita ferruginosa koreana Habe et Ito.
Habe, 1977: 65 (cited as of 1965a).
“Shells of the World in Colour, Vol. Il.
The Tropical Pacific.” Tadashige Habe and
Sadao Kosuge
First published January 15, 1966 (1966a),
this work preceded an article in Venus by the
same authors (1966b) in which new taxa, first
appearing in Volume ll, are proposed. There
is no indication in Volume II that these taxa
are newly introduced therein. The authors
stated (1966b) that these “genera and spe-
cies were figured and briefly described” in
1966a and that “they are redescribed in de-
tail herewith in the nomenclatural value.” Dr.
Kosuge (personal comm., March 15, 1995)
has confirmed that the genera all must date
from the Venus article.
Dr. Kosuge also advises that there are at
least ten printings of this work, the 10th ap-
pearing in March, 1991.
Printings that we have seen:
Printing 1 January 15, 1966 (1966a)
2 November 1, 1966 (1966c)
3 February 1, 1972
5 November 11, 1974
6
September 1, 1976
The following new species appear (using
the original arrangement of families). Impor-
tant changes between printings and refer-
ences from other sources are also listed.
Gastropoda
Acmaeidae
(32) Patelloida (Collisellina) saccharinoides
Habe & Kosuge, 1966a
Patelloida (Collisellina) saccharinoides Habe
et Kosuge. Habe & Kosuge, 1966a: 6, pl.
2, fig. 10; 1966c, 1972, 1974, 1976: ibid.
Patelloida (Collisellina) saccharinoides Habe
et Kosuge (sp. nov.). Habe & Kosuge,
1966b: 312.
Patelloida (Collisellina) saccharioides [sic]
Habe et Kosuge (sp. nov.). Habe & Ko-
suge, 1966b: 326, pl. 29, fig. 6 (this spell-
ing also on plate caption on same page).
Patelloida (Collisellina) saccharinoides Habe
et Kosuge. Habe, 1977: 103 (cited as of
1966a).
Turbinidae
(33) Astralium yamamurae Habe & Kosuge,
1966a
Astralium yamamurai [sic] Habe et Kosuge.
Habe & Kosuge, 1966a: 11; 1966c: ibid.
(error in spelling corrected on page 121
and in all later usages)
Astralium (Distellifer [sic] yamamurae Habe
et Kosuge. Habe & Kosuge, 1966a: 121,
pl. 45, fig. 11; 1966c, 1972, 1974, 1976:
ibid.
Astralium yamamurae Habe et Kosuge. Habe
& Kosuge, 1972: 11; 1974, 1976: ibid.
Astralium (Destellifer) yamamurae Habe et
Kosuge (sp. nov.) Habe & Kosuge,
1966b: 313, 327 (with reference to
1966a, pl. 45, fig. 4 [sic; error for fig. 11]).
Astralium (Destellifer) yamamurae Habe et
Kosuge. Habe, 1977: 133 (cited as of
1966a; 1966b cited as “name only’).
Littorinidae
(34) Granulilittorina Habe & Kosuge, 1966b
(35) Granulilittorina philippiana Habe & Ko-
suge, 1966a
Granulilittorina philippiana Habe et Kosuge.
Habe & Kosuge, 1966a: 20, pl. 6, fig. 13;
1966c; ibid. (genus-group name =
nomen nudum).
Granulilittorina philippiana Habe et Kosuge
(gen. et sp. nov.). Habe & Kosuge,
1966b: 313, 328 (with reference to
1966a, pl. 6, fig. 13).
42 PENITT&:BIEEER
Granulilittorina millegrana (Philippi) Habe et
Kosuge. Habe & Kosuge, 1972, 1974,
1976: 20, pl. 6, fig: 13:
Granulilittorina Habe et Kosuge. Habe, 1977:
45. (cited as of 1966b, with type, by
monotypy, G. philippiana Habe & Ko-
suge; 1966a not mentioned).
Granulilittorina philippiana Habe et Kosuge.
Habe, 1977: 96 (cited as of 1966a;
stated to be a synonym of G. millegrana
(Philippi, 1848)).
Taxonomic note: In 1966a no indication was
given that this was a newly introduced
genus-group name. Rosewater (1970:
491-493) used Granulilittorina as a valid
subgenus of Nodilittorina. In treating the
genus-group name he cited both 1966a
and 1966b. However, under the species
name (in the synonymy of N. (G.) mille-
grana) he listed as of 1966b with “Tfig-
ured in] Habe and Kosuge” 1966a
(square brackets in quote are of Rose-
water).
Cerithiidae
(36) Clypeomorus batillariaeformis Habe &
Kosuge, 1966a
Clypeomrus [sic] batillariaeformis Habe et
Kosuge. Habe & Kosuge, 1966a: 23, pl.
7, fig. 14; 1966c: ibid.
Clypeomorus batillariaeformis Habe et Ko-
suge (sp. nov.) Habe & Kosuge, 1966b:
314, 328, pl. 29, fig. 13 (with reference to
1966a, pl. 7, fig. 14).
Clypeomorus batillariaeformis Habe et Ko-
suge. Habe & Kosuge, 1972, 1974, 1976:
23, pl. 7, fig. 14.
Clypeomorus batillariaeformis Habe et Ko-
suge. Habe, 1977: 26 (cited as of 1966a;
original misspelling of genus shown and
corrected).
Taxonomic note: Houbrick (1985: 51) treated
this species in detail and attributed it to
Habe & Kosuge, 1966b, without any
mention of 1966a.
Cypraeidae
(37) Ficadusta Habe 8 Kosuge, 1966b
Ficadusta pulchella (Swainson, 1823). Habe
8 Kosuge, 1966a: 40, pl. 14, figs. 15, 16;
1966c: ibid. (genus-group name =
nomen nudum).
Ficadusta Habe et Kosuge (gen. nov.). Habe
8 Kosuge, 1966b: 314, 329 (with refer-
ence to 1966a; type species: Cypraea
pulchella Swainson).
Ficadusta pulchella (Swainson). Habe 8 Ko-
suge, 1966b: 326 (plate expl.), pl. 29,
figs: 11,112:
Ficadusta pulchella (Swainson, 1923 [sic]).
Habe 8 Kosuge, 1972: 40, pl. 14, figs.
15, 16; 1974, 1976: ibid. (type reset in
1972 to correct English common name).
Ficadusta Habe et Kosuge, 1966. Habe,
1977: 40 (cited as of 1966b, but with
type by “monotypy,” whereas in 1966b
it was designated; 1966a listed as
“name only”).
Cymatiidae
(38) Reticutriton Habe & Kosuge, 1966b
Reticutriton pfeifferianum (Reeve, 1844).
Habe & Kosuge, 1966a: 43, pl. 15, fig.
14; 1966c: ibid. (genus-group name =
nomen nudum).
Reticutriton Habe et Kosuge (gen. nov.).
Habe & Kosuge, 1966b: 315, 330 (with
reference to 1966a; type species: Triton
pfeifferianus Reeve).
Reticutriton pfeifferianus (Reeve, 1844). Habe
& Kosuge, 1972: 43, pl. 15, fig. 14; 1974,
1976: ibid.
Reticutriton Habe et Kosuge. Habe, 1977:
102 (cited as of 1966b; 1966a not men-
tioned).
Muricidae
(39) Spinidrupa Habe & Kosuge, 1966b
Spinidrupa eurantha [sic] (A. Adams). Habe &
Kosuge, 1966a: 54, pl. 20, fig. 4; 1966c,
1972, 1974, 1976: ibid. (genus-group
name = nomen nudum).
Spinidrupa Habe et Kosuge (gen. nov.). Habe
& Kosuge, 1966b: 315, 330 (with refer-
ence to 1966a; type species: Murex
eurantha [sic] A. Adams [p. 315; as eu-
racantha on p. 330; = Murex euracanthus
A. Adams, 1851].
Spinidrupa Habe et Kosuge. Habe, 1977: 115
(cited as of 1966b; 1966a listed as
“name only”).
Pyrenidae (Columbellidae)
(40) Pyrene testudinaria nigropardalis Habe &
Kosuge, 1966a
Pyrene testudinalia [sic] nigropardalis Habe et
SHELLS OF THE WORLD IN COLOR 43
Kosuge. Habe & Kosuge, 1966a: 57, pl.
21, fig. 3; 1966c: ibid.
Pyrene testudinaria nigropardalis Habe et Ko-
suge (sp. nov.). Habe & Kosuge, 1966b:
316, 331, pl. 29, fig. 7 (with reference to
1966a).
Pyrene testudinaria nigropardalis Habe et Ko-
suge. Habe 8 Kosuge, 1972, 1974, 1976:
57, Pl 21, 19: 3.
Pyrene testudinalia [sic] nigropardalis Habe et
Kosuge. Habe, 1977: 83 (cited as of
1966a).
(41) Pyrene lacteoides Habe & Kosuge,
1966a
Ругепе lacteoides Habe et Kosuge. Habe 8
Kosuge, 1966a: 57, pl. 21, fig. 8; 1966c,
1972, 1974, 1976: ibid.
Pyrene lacteoides Habe et Kosuge (sp. nov.).
Habe & Kosuge, 1966b: 316, 330, pl. 29,
fig. 8 (with reference to 1966a).
Pyrene lacteoides Habe et Kosuge. Habe,
1977: 68 (cited as of 1966a).
Nassariidae
(42) Plicarcularia gibbosuloidea Habe & Ko-
suge, 1966a
Pliarcularia [sic] gibbosuloidea Habe et Ko-
suge. Habe & Kosuge, 1966a: 60, pl. 22,
figs. 5, 6; 1966c, 1972, 1974, 1976: ibid.
Pliarcularia [sic] gibbosuloidea Habe et Ko-
suge (sp. nov.). Habe & Kosuge, 1966b:
317,
Plicarcularia gibbosuloidea Habe et Kosuge
(sp. nov.). Habe & Kosuge, 1966b: 326
[pl. explanation], 331, pl. 29, figs. 2, 3
(with reference to 1966a).
Plicarcularia gibbosuloidea Habe & Kosuge.
Habe, 1977: 44 (cited as of 1966a; orig-
inal misspelling of genus shown and cor-
rected).
Galeoidae (Galeolidae in 1966a: 64 and
1966c: 64; correct on p. 65 and in later
printings)
(43) Hemifusus carinifer Habe & Kosuge,
1966a
Hemifusus carinifera [sic] Habe et Kosuge.
Habe & Kosuge, 1966a: 64, pl. 23, fig. 2;
1966c, 1972, 1974, 1976: ibid.
Hemifusus cariniferus Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 317, 332,
pl. 29, fig. 17 (with reference to 1966a).
Hemifusus cariniferus Habe et Kosuge. Habe,
1977: 29 (cited as of 1966a).
Note: Originally introduced as an adjective in
the female form, the ending has to be
adjusted to the masculine -fer (-fer, -fera,
-ferum, meaning “bearing””; as opposed
to -ferus-a-um, meaning ‘‘wild’’).
Fasciolariidae
(44) Latirus stenomphalus Habe 8 Kosuge,
1966a
Latirus stenomphalus Habe et Kosuge. Habe
8 Kosuge, 1966a: 68, 122, pl. 45, fig. 16
(with reference to Kira, [1954]: pl. 30, fig.
16, which is the species Kira figured as
Latirus recurvirostrum Schubert 8 Wag-
ner); 1966a, 1972, 1974, 1976: ibid.
Latirus stenomphalus Habe et Kosuge (sp.
nov.). Habe 8 Kosuge, 1966b: 318, 334,
(with reference to Latirus recurvirostrum
Kira, 1954: pl. 30, fig. 16 [on p. 318] and
to 1966a [p. 334]; misspelled sttnom-
phalus on p. 318). This reference to Kira
is to the species he figured as Latirus
recurvirostrum Schubert & Wagner.
Latirus stenomphalus Habe et Kosuge. Habe,
1977: 116 (cited as of 1966a).
Mitridae
(45) Vexillum rubrocostatum Habe & Kosuge,
1966a
Vexillum rubrocostatum Habe et Kosuge.
Habe & Kosuge, 1966a: 73, pl. 28, fig. 9;
1966c, 1972, 1974, 1976: ibid.
Vexillum rubrocostatum Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 319, 333,
pl. 29, fig. 4 (with reference to 1966a).
Vexillum rubrocostatum Habe et Kosuge.
Habe, 1977: 102 (cited as of 1966a).
(46) Nebularia yaekoae Habe & Kosuge,
1966a
Nebularia yaekoae Habe et Kosuge. Habe &
Kosuge, 1966a: 76, pl. 28, fig. 34; 1966c,
1972, 1974, 1976: ibid.
Nebularia yaekoae Habe et Kosuge (sp. nov.).
Habe & Kosuge, 1966b: 319, 333, pl. 29,
fig. 10 (with reference to 1966a).
Nebularia yaekoae Habe et Kosuge. Habe,
1977: 131 (cited as of 1966a).
Harpidae
(47) Harpa kawamurai Habe, in Habe & Ko-
suge, 1972
Harpa striata (Lamarck, 1816). Habe & Ko-
44 PETIT & BIELER
suge, 1966a: 79, pl. 30, fig. 2; supple-
mental pl. 1, fig. 2; 1966c: ibid.
Harpa kawamurai Habe. Habe, in Habe & Ko-
suge, 1972: 79, pl. 30, fig. 2; supplemen-
tal pl. 1, fig. 2; 1974, 1976 (no indication
that name is new).
Harpa kawamurai Habe & Kosuge, 1973 [sic].
Habe, 1975b: 10 (listed as “invalid” and
as ‘= Harpa major Röding, 1798”).
Harpa kawamurai Habe & Kosuge, 1973 [sic].
Matsukuma & Okutani, 1986: 6.
Taxonomic note: The 3rd printing of Habe &
Kosuge, where this species first ap-
pears, is rare, and we have located only
one copy. Not listed by Habe (1977). The
Japanese text of Habe (1975b: 10)
states that, according to personal com-
munication with Dr. Rehder, this nominal
species is a form of Harpa major Róding,
1798.
(48
—
Harpa kajiyamai Habe, in Habe & Ko-
suge, 1972
Награ cancellata (Roding, 1798). Habe &
Kuroda, 1966a: 79, pl. 30, fig. 3; supple-
mental pl. 1, fig. 3; 1966c: ibid.
Harpa kajiyamai Habe. Habe & Kosuge, 1972:
79, pl. 30, fig. 3; supplemental pl. 1, fig.
3
Harpa kajiyamai Rehder, 1973: 244, pl. 188,
figs. 3, 4 (described from specimens re-
ceived from Habe, who was stated to
have recognized the species as new and
given it a provisional name, and re-
quested that it be named for the collec-
tor).
Harpa kajiyamai Rehder. Habe & Kosuge,
1974: 79, pl. 30, fig. 3; supplemental pl.
1, fig. 3; 1976: ibid.
Taxonomic note: Walls (1980: 191) in his list
of Harpa species includes both H. kajiy-
amai Habe, 1970 [sic], and H. kajiyamai
Rehder, 1973, indicating that both are ‘‘=
[Harpa] harpa,” a synonymy we do not
endorse. This species name must be at-
tributed to Habe (1972). Not listed by
Habe (1977).
Volutidae
(49) Volutoconus grossi mcmichaeli Habe &
Kosuge, 1966a
Volutoconus grossi mcmichaeli Habe & Ko-
suge. Habe & Kosuge, 1966a: 86, pl. 33,
fig. 1; 1966c, 1972, 1974, 1976: ibid.
Volutoconus grossi mcmichaeli Habe et Ko-
suge (sp. nov.). Habe & Kosuge, 1966b:
320, 335, pl. 29, fig. 19 (with reference to
1966a).
Volutoconus grossi mcmichaeli Habe et Ko-
suge. Habe, 1977: 74 (cited as of 1966a).
Turridae
(50) Brachytoma kurodai Habe & Kosuge,
1966a
Brachytoma kurodai Habe et Kosuge. Habe &
Kosuge, 1966a: 96, pl. 38, fig. 13; 1966c,
1972, 1974, 1976: ibid.
Brachytoma kurodai Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 320, 335,
pl. 29, fig. 14 (with reference to 1966a)
Brachytoma kurodai Habe et Kosuge. Habe,
1977: 66 (cited as of 1966a).
(51) Brachytoma kawamurai Habe & Kosuge,
1966a
Brachytoma kawamurai Habe et Kosuge.
Habe & Kosuge, 1966a: 96, pl. 38, fig.
14; 1966c, 1972, 1974, 1976: ibid.
Brachytoma kawamurai Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 321, 336,
pl. 29, fig. 9 (with reference to 1966a)
Brachytoma kawamurai Habe et Kosuge.
Habe, 1977: 58 (cited as of 1966a).
(52) Brachytoma vexillium Habe & Kosuge,
1966a
Brachytoma vexillium Habe et Kosuge. Habe
& Kosuge, 1966a: 96, pl. 38, fig. 15;
1966c, 1972, 1974, 1976: ibid.
Brachytoma vexillum Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 321, 336,
pl. 29, fig. 5 (with reference to 1966a).
Brachytoma vexillum Habe et Kosuge. Habe,
1977: 130 (cited as of 1966a; original
spelling not mentioned).
Taxonomic note: The original spelling of the
specific name, although obviously a mis-
spelling or typographical error, must be
retained in accordance with ICZN Code
Article 32.
Epitoniidae
(53) Eglisia brunnea Habe & Kosuge, 1966a
Eglisia brunnea Habe et Kosuge. Habe & Ko-
suge, 1966a: 103, pl. 40, fig. 16; 1966c:
ibid.
Eglisia brunnea Habe et Kosuge (sp. nov.).
Habe & Kosuge, 1966b: 322, 337, pl. 29,
fig. 18 (with reference to 1966a).
SHELLS OF THE WORLD IN COLOR 45
Eglisia lanceolata brunnea Habe et Kosuge.
Habe & Kosuge, 1972: 103, pl. 40, fig.
16; 1974, 1976: ibid.
Eglisia brunnea Habe et Kosuge. Habe, 1977:
28 (cited as of 1966a).
Bivalvia
Limidae
(54) Mantellum perfragile Habe 8 Kosuge,
1966a
Mantellum perfragile Habe et Kosuge. Habe
8 Kosuge, 1966a: 144, 177, pl. 68, fig. 6;
1966c, 1972, 1974, 1976: 144.
Mantellum perfragile Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 323, 338.
(not figured; 1966a not referred to)
Limaria perfragile Habe et Kosuge. Habe 4
Kosuge, 1972, 1974, 1976: 177, pl. 68,
fig. 6 (as Mantellum on p. 144).
Mantellum perfragile Habe et Kosuge. Habe,
1977: 95 (cited as of 1966a and placed in
Limaria (Platilimaria)).
Ostreidae
(55) Anomiostrea Habe & Kosuge, 1966b
Anomiostrea pyxidata (Adams et Reeve,
1850). Habe & Kosuge, 1966a: 144, pl.
55, fig. 9; 1966c, 1972, 1974, 1976: ibid.
(genus-group name = nomen nudum).
Anomiostrea Habe et Kosuge (gen. nov.).
1966b: 323, 338, with type designated
as Ostrea pyxidata Adams et Reeve (with
reference to 1966a).
Anomiostrea Habe et Kosuge. Habe, 1977:
23 (cited as of 1966b; 1966a not men-
tioned).
Taxonomic note: Listed under “nomina du-
bia” by Stenzel (1971: N1167, figs.
J140a-c), who also showed the name of
the type species to be preoccupied.
Type species renamed Anomiostrea cor-
alliophila Habe, 1975a (new name for O.
pyxidata Adams & Reeve, 1848 [sic; =
1850] non Born, 1780 [sic; = 1778].
Cardiidae
(56) Laevicardium rubropictum Habe & Ko-
suge, 1966a
Laevicardium rubropictum Habe et Kosuge.
1966a: 153, pl. 59, fig. 2; 1966c, 1972,
1974, 1976: ibid.
Laevicardium rubropictum Habe et Kosuge
(sp. nov.). 1966b: 324, 339, pl. 29, fig. 20
(with reference to 1966a)
Laevicardium rubropictum Habe et Kosuge.
Habe, 1977: 102 (cited as of 1966a).
(57) Vasticardium nigropunctatum Habe &
Kosuge, 1966a
Vasticardium nigropunctatum Habe et Ko-
suge. Habe & Kosuge, 1966a: 154, pl.
59, fig. 9; 1966c, 1972, 1974, 1976: ibid.
Vasticardium nigropunctatum Habe et Ko-
suge (sp. nov.). Habe & Kosuge, 1966b:
324, 340, pl. 29, fig. 16 (with reference to
1966a).
Vasticardium nigropunctatum Habe et Ko-
suge. Habe, 1977: 84 (cited as of 1966a).
Mactridae
(58) Macrotoma yamamurae Habe & Kosuge,
1966a
Mictrotoma [sic] yamamurae Habe et Ko-
suge. Habe & Kosuge, 1966a: 166, pl.
65, fig. 8; 1966c: ibid.
Mactrotoma yamamurae Habe et Kosuge (sp.
nov.). Habe & Kosuge, 1966b: 325, 340,
pl. 29, fig. 15 (with reference to 1966a;
original misspelling of genus noted).
Mactrotoma yamamurae Habe et Kosuge.
Habe & Kosuge, 1972: 166, pl. 65, fig. 8.
Heterocardia gibbosula Philippi [sic; = De-
shayes]. Habe & Kosuge, 1974: 166, pl.
65, fig. 8; 1976: ibid.
Mactrotoma yamamurae Habe et Kosuge.
Habe, 1977: 133 (cited as of 1966a;
stated to be a synonym of Heterocardia
gibbosula Deshayes, 1855).
ACKNOWLEDGEMENTS
The following made copies of publications
available or otherwise responded to our re-
quests for data: Dr. E. V. Coan, Dr. В. М.
Kilburn, Dr. H. G. Lee, Dr. J. H. McLean, Mr.
Thomas C. Rice, Dr. Gary Rosenberg, Mr.
Walter Sage, and Dr. Emily H. Vokes. Dr.
Sadao Kosuge corresponded with us con-
cerning later printings of both volumes and
the availability of the taxa. Dr. H. D. Cameron,
University of Michigan, provided etymologi-
cal advice. We are especially indebted to Dr.
Takahiro Asami, Tachikawa College of To-
kyo, whose translations from the Japanese
helped us in deciding on the validity of taxon
46 PETIT & BIELER
descriptions, and to Dr. M. G. Harasewych
who, while in Japan, searched for and ob-
tained for us a copy of the elusive 3rd printing
of Habe & Kosuge. We also wish to thank two
anonymous reviewers for their comments.
LITERATURE CITED
BIELER, R. & R. E. PETIT, 1990, On the various
editions of Tetsuaki Kira’s “Coloured illustra-
tions of the shells of Japan” and “Shells of the
western Pacific in color Vol. 1,” with an anno-
tated list of new names introduced. Malacologia
32: 131-145.
HABE, T., 1975a, New name for Anomiostrea pyx-
idata (Adams & Reeve) (Ostreidae). Venus 33:
184 (April).
HABE, T., ed., 1975b, Publication for commemo-
rate 77th anniversary of the birth of Mr. Ryosuke
Kawamura. Illustration of shells described by and
dedicated to Mr. R. Kawamura. 20 pp., incl. 5
pls. Tokyo (December).
HABE, T., 1977, Catalogue of molluscan taxa de-
scribed by Tadashige Habe during 1939-1975,
with illustrations of hitherto unfigured species
(for commemoration of his sixtieth birthday). 185
pp. incl. 7 pls.; Tokyo. [compiled by T. Inaba and
K. Oyama, but authorship credited to Habe on
page 2]
HABE, T. & K. ITO, 1965a, Shells of the world in
colour, Vol. |. The northern Pacific. viii, [2 pp.
map], 176 pp., 56 pls.; Hoikusha, Osaka [addi-
tional printings listed in this paper].
HABE, T. & K. ITO, 1965b, New genera and spe-
cies of shells chiefly collected from the North
Pacific. Venus 24: 16-45, pls. 2-4 (July 31).
HABE, T. & K. ITO, 1968, Buccinid species from
Rausu, Hokkaido. Venus 27: 1-8, pl. 1 (August
31).
HABE, T. 4 S. KOSUGE, 1966a, Shells of the world
in colour, Vol. |. The tropical Pacific. vii, [2 pp.
map], 193 pp., pls. 1-68, supplemental pls. 1-2;
Hoikusha, Osaka (January 15; additional print-
ings listed in this paper).
HABE, T. & S. KOSUGE, 1966b, New genera and
species of the tropical and subtropical Pacific
molluscs. Venus 24: 312-341, pl. 29 (May 17).
HOUBRICK, R.S., 1985. Genus Clypeomorus
Jousseaume (Cerithiidae: Prosobranchia).
Smithsonian Contributions to Zoology 403:
1-131.
KIRA, T., 1954. [Coloured illustrations of the shells
of Japan]. [viii] + 172 + 24 pp., 67 pls; Hoikusha,
Osaka (additional printings listed in Bieler 8
Petit, 1990).
KURODA, T., T. HABE & K. OYAMA, 1971. The sea
shells of Sagami Bay. Maruzen, Tokyo. xix + 741
pp. [in Japanese], pls. 1-121, 489 pp. [in En-
glish], 51 pp. index, map.
MATSUKUMA, A. & T. OKUTANI, 1986. Studies on
the Kawamura collection (Mollusca) in the Na-
tional Science Museum, Tokyo-ll. Catalogue of
type specimens, with description of Pinna cello-
phana n. sp. (Bivalvia). Venus 45: 1-10
REHDER, H. A., 1973, The family Harpidae of the
world. Indo-Pacific Mollusca 3: 207-274.
ROSEWATER, J., 1970, The family Littorinidae in
the Indo-Pacific. Part I. The subfamily Littorini-
nae. Indo-Pacific Mollusca 2: 417-528.
STENZEL, H. B., 1971, Oysters. Treatise on Inver-
tebrate Paleontology, Part N, Volume 3, Mol-
lusca 6, Bivalvia. Pp. N953-N1224.
WALLS, J. G., 1980, Conchs, tibias and harps.
T.H.F. Publications Inc. Ltd., Neptune, New Jer-
sey. 191 pp.
Revised Ms. accepted 28 November 1995
MALACOLOGIA, 1996, 38(1-2): 47-58
ULTRASTRUCTURAL STUDY OF EUSPERMIOGENESIS IN CLYPEOMORUS
BIFASCIATA AND CLYPEOMORUS TUBERCULATUS (PROSOBRANCHIA:
CERITHIIDAE) WITH EMPHASIS ON ACROSOME FORMATION
Fadwa A. Attiga' & Hameed A. Al-Hajj
Department of Biological Sciences, University of Jordan, Amman, Jordan
ABSTRACT
The ultrastructure of euspermiogenesis and euspermatozoa of Clypeomorus bifasciata and
C. tuberculatus are almost identical. Early spermatids have oval to spherical nuclei, sparse
endoplasmic reticulum, few mitochondria, and a well-developed Golgi complex with many
vesicles in its vicinity. Acrosome differentiation occurs anywhere within the cytoplasm, and
begins with a proacrosomal vesicle, which becomes cup-shaped and plugged at its edges with
a dense interstitial granule. Microtubules are embedded in the matrix between the outer and
inner acrosomal membranes. The acrosomal vesicle becomes aligned parallel to the antero-
posterior nuclear axis, and changes into an inverted flask shape, with two external supporting
structures at its basal margins. The interstitial granule becomes hat-shaped, separating the
acrosome from the nucleus. The mature acrosome consists of a flat cone with microtubules in
its core, an acrosomal rod-like material, and a basal plate. Nuclear shape changes from spher-
ical to hammer-head to club-shape, with a posterior invagination enclosing the initial axonemal
portion. The fine chromatin material of early spermatids changes to fibrillar, lamellar, and finally
very compact material. The euspermatozoan midpiece originates from fusion of spermatid
mitochondria into four large spheres, which are later organized into four non-helical mitochon-
drial elements, two of which are large and the other two are extremely small. A dense ring
structure marks the junction between the midpiece and the glycogen piece. The latter consists
of nine tracts of glycogen granules surrounding nine axonemal doublets. The results of this
study suggest that acrosomal ultrastructure could be used to establish phylogenetic relation-
ships in Cerithiacea at the generic level.
INTRODUCTION
Morphological diversity of spermatozoa in
prosobranchs, as in other animal groups, has
been considered as a tool that can be used to
ascertain evolutionary paths, through building
up phylogenetic and taxonomic. affinities
among species (Franzen, 1955, 1956, 1970;
Nishiwaki, 1964; Healy, 1983a, 1988a; Koike,
1985). Based on ultrastructural studies of
spermiogenesis and sperm morphology, me-
sogastropods as a part of caenogastropods
(mesogastropods and neogastropods) are
classified into two groups. Members of the
first group have short nuclei with shallow
basal invaginations, associated with conical
or flattened acrosomes. The midpiece may
show modification of cristae into parallel cri-
stal plates, and it is separated from the gly-
cogen piece by a dense ring structure. The
glycogen piece consists of axonemal micro-
tubules and nine tracts of glycogen granules,
whereas the short end piece is composed of
an axoneme surrounded only by a plasma
membrane. This group of caenogastropods
includes superfamilies Cerithiacea (Healy,
1982a, b, 1983a; Afzelius & Dallai, 1983;
Koike, 1985), Viviparacea (Griffond, 1980;
Koike, 1985), and Cyclophoracea (Selmi &
Giusti, 1980; Healy, 1984; Kohnert & Storch,
1984a, b, Koike, 1985). All other superfamilies
in Caenogastropoda are classified into the
second group, which shares with the first
group similar glycogen pieces, dense ring
structures and end pieces. On the other hand,
members of this group have apical acrosomal
vesicles and accessory acrosomal mem-
branes, whereas their short or long tubular
nuclei may be completely invaginated by the
axoneme (Healy, 1988a). The midpiece ele-
ments are helically coiled, with usually un-
modified cristae (Healy, 1983a, 1986b; Max-
“This work was conducted as part of Fadwa Attiga’s Master thesis. The George Washington University, Columbian College
and Graduate School of Arts and Sciences, Department of Biological Sciences, Ph.D. Program. Author to whom all
correspondence should be mailed. Address: 2301 E St. NW, Apt # A406, Washington, DC 20037, U.S.A.
48 ATTIGA & AL-HAJJ
well, 1983; Kohnert & Storch, 1984a; Koike,
1985; Jaramillo et al., 1986). Furthermore,
comparative sperm ultrastructure has been
useful in establishing the affinities of many
cerithiacean superfamilies of the Caenogas-
tropoda (Healy, 1982a, b, 1983a, 1986a, b,
1988a, b, 1990a, b, 1993; Houbrick, 1988).
The present work deals with the ultrastruc-
ture of euspermiogenesis and mature eu-
sperm (typical sperm) in two species of the
superfamily Cerithiacea (family Cerithiidae)
that inhabit the rocky shore of the Gulf of
Aqaba (Houbrick, 1985; Hulings, 1986).
These are: Clypeomorus bifasciata (Sowerby,
1855) [= С. moniliferum (Kiener, 1841),
auett.], and C. tuberculatus (Linnaeus, 1758)
[= C. petrosa gennesi (Fisher & Vignal, 1901)].
Comparative study of spermiogenesis and
sperm morphology of the two cerithiid spe-
cies as well as other reported cerithiids aims
to emphasize species-specific characters
between cerithiids from different geographi-
cal regions, and to establish the phylogenetic
status of cerithiaceans among prosobranchs.
MATERIALS AND METHODS
Specimens were collected monthly for a
year in the intertidal zone opposite to the Ma-
rine Science Station of the Gulf of Aqaba. The
shell was gently broken, and the testis, re-
moved by dissection, was immediately im-
mersed in 2.5% glutaraldehyde in filtered sea
water for 2 hours at room temperature. The
tissue was rinsed thoroughly in filtered sea
water, post fixed in 1% OsO, solution in fil-
tered sea water, dehydrated in acetone and
embedded in Spurr's (1969) medium. Blocks
were cut with Sorval MT 2B ultramicrotome
using glass knives, and ultrathin sections
(50-60 nm) were stained with uranyl acetate
and lead citrate. Electron microscopic exam-
inations were done with a Zeiss EM 10B
transmission electron microscope operated
at 60 KV.
RESULTS
The various stages of euspermiogenesis in
Clypeomorus bifasciata and C. tuberculatus
are almost identical. Therefore, the following
description applies for both species unless
otherwise mentioned.
Early spermatids are spherical to ovoidal,
with eccentric nuclei. The chromatin material
is granular, with some local aggregations of
no specific pattern. The granular cytoplasm
contains few cisternae of endoplasmic retic-
ulum, few mitochondria, and a well-devel-
oped Golgi complex with many vesicles at
the extremities of its cisternae, indicating ac-
tivity (Fig. 1). Nutritive cells can be seen in the
intercellular space with many elongated
pseudopodia (Fig. 1).
Acrosome development can be divided
into two major phases; the pre-attachment
acrosome and the post-attachment one, in
reference to its attachment to the nucleus.
Acrosomal genesis during the first phase be-
gins with a single proacrosomal vesicle as-
sociated with Golgi complex, in addition to
many nearby dense vesicles that are likely to
be utilized in the production of the acrosomal
elements (Fig. 2). Later, this vesicle attains an
inverted U-shape due to posterior indenta-
tion, and a dense interstitial granule plugs the
prospective subacrosomal space (Figs. 3, 4).
The dispersion of dense material from this
granule and its deposition on the inner and
outer acrosomal membranes assist in the ac-
centuation of the acrosome (Figs. 4, 9). Two
dense internal supporting structures appear
within the acrosomal body, and microtubules
constitute the skeleton of the acrosomal
cone between the inner and outer acrosomal
membranes (Figs. 4, 8, 9).
The second phase of acrosomal develop-
ment is demarcated by the attachment of the
basal interstitial granule to a depression on
the anterior pole of the fibrous nucleus, op-
posite to the site of axoneme development
(Fig. 10). The acrosome rotates 90” to be-
come aligned parallel to the antero-posterior
axis of the developing spermatid (Figs. 10,
11). Following its attachment, the acrosome
looks like an inverted flask due to a constric-
tion at its posterior part (Figs. 11, 12). Two
crescent-shaped external supporting struc-
tures can be seen at the basal margins of the
acrosome near its attachment point to the
nucleus. The post-attachment acrosome is
further elongated, while the dense interstitial
granule gives rise to a basal plate between
the acrosome and the nucleus (Figs. 11, 12).
The acrosome of the mature euspermato-
zoon in C. bifasciata and C. tuberculatus con-
sists of three structures; acrosomal cone, ac-
rosomal rod-like material, and basal plate
(Figs. 16 inset, 17). The tapering cone may
occasionally show parallel plate-like sub-
structures, and it is characterized by basal
ULTRASTRUCTURAL STUDY OF EUSPERMIOGENESIS 49
e
FIG. 1. Clypeomorus bifasciata. Early spermatid showing nucleus (N), mitochondrion (M), Golgi complex
(GC) and associated vesicles (V). Notice pseudopodia (PP) of the nutritive cell (NC). x11,500
FIG. 2. C. bifasciata. Early spermatid showing peripheral chromatin lining the nucleus (N), Golgi complex
(GC), and interstitial granule (IG). x31,250
50 ATTIGA & AL-HAJJ
FIG. 3. C. bifasciata. Early spermatid showing nucleus (N), two mitochondrial (M) spheres at nuclear base,
Golgi complex (GC), vesicles (V), and differentiating proacrosomal vesicle (PAV). Notice cytoplasmic bridge
(asterisk). x16,000
FIG. 4. C. bifasciata early spermatid. Section showing a differentiating acrosome with microtubules (MT) in
its cone, internal supporting structures (arrows) subacrosomal space (SAS), and interstitial granule (IG).
x36,000
FIG. 5. C. tuberculatus early spermatid. Section showing nucleus (N), with sites of the attachment of
mitochondrial (M) spheres (arrows). x15,200
FIG. 6. C. tuberculatus early spermatid. Section showing nuclear (N) base with mitochondrial (M) spheres,
implantation fossa (IF), centriolar derivative (CD), and axoneme (AX). x56,700
FIG. 7. C. bifasciata early spermatid. Section showing four mitochondrial (M) spheres surrounding the
axoneme (AX) as the first stage of midpiece development. x15,000
ULTRASTRUCTURAL STUDY OF EUSPERMIOGENESIS 51
bulges in the cone wall that cause a constric-
tion in the subacrosomal space. The latter,
which extends the whole length of the ac-
rosomal cone, contains an acrosomal rod-
like material (Figs. 16 inset, 17). À dense
basal plate linking the acrosome and the nu-
cleus can be seen as a straight dense layer
between the two structures. Cross sections
in the mature acrosome indicate its flatness,
and microtubules assume a zipper-like struc-
ture in the matrix between the inner and outer
acrosomal membranes (Fig. 18).
Chromatin condensation starts with the
formation of a uniformly thick layer at the pe-
riphery of the nucleus (Figs. 2, 3). As spermi-
ogenesis proceeds, the granular chromatin
accumulates at the posterior nuclear pole,
and the nucleus undergoes antero-posterior
compression, leading to gradual increase in
the nuclear width at the expense of its length
(Fig. 8). In addition, the axoneme extends
backwards from a centriolar derivative in the
implantation fossa, so that vertical sections
through a developing euspermatozoon at this
stage present hammer-head and handle con-
figurations (Fig. 10).
A second phase of chromatin condensa-
tion is evident in middle spermatids as the
mid-anterior portion of the nucleus, opposite
to the axoneme, begins a forward movement.
During this phase, fibrils are arranged longi-
tudinally parallel to the nuclear antero-poste-
rior axis (Fig. 11). As fibrils increase in thick-
ness, they stan to fuse into fibers and
subsequently into lamellae representing
thereby the lamellar phase of chromatin con-
densation (Figs. 13-15). Chromatin conden-
sation culminates in a homogenous, com-
pact club-like nucleus with no distinct
ultrastructure (Fig. 19).
Concomitant with nuclear condensation, a
growing axoneme pushes the nucleus for-
ward to increase its length, while the nuclear
width is reduced under a force of lateral com-
pression. This leads to progressive lengthen-
ing of the antero-posterior nuclear axis, thus
reversing its trend in the previous stages
(Figs. 10, 11). The mature nucleus in C. bifas-
ciata and C. tuberculatus has a short poste-
rior invagination accommodating the proxi-
mal portion of the axoneme (Figs. 16, 19).
The euspermatozoan tail in C. bifasciata
and C. tuberculatus is composed of a middle
piece, a glycogen piece and an end piece.
The posterior nuclear envelope becomes in-
dented at its center defining thereby the im-
plantation fossa, which represents the point
of axoneme development (Fig. 6). The gene-
sis of the axoneme appears to be associated
with a single dense structure (centriolar de-
rivative), which does not seem to possess the
common pattern of centriolar arrangement
(Fig. 6). As nuclear condensation com-
mences, mitochondrial fusion gives rise to
four large spherical mitochondria at the pos-
terior nuclear pole (Fig. 7). The association of
these spheres with the nucleus is achieved
by their attachment to four posterior nuclear
depressions, and it represents the first step
in midpiece development, which is concom-
itant with the granular phase of nuclear con-
densation. The mitochondrial cristae are
modified into parallel cristal plates that have
undergone considerable reorganization as
mitochondria form a sheath around the typi-
cal 9 + 2 axoneme (Figs. 10, 11). Transverse
sections through the midpiece reveal four
non-helically arranged mitochondrial ele-
ments, two of which are semicircular large
elements that are arranged perpendicular to
the central pair of axonemal microtubules,
and each reveals multiple cristal plates. The
other two mitochondrial elements are ex-
tremely small and are aligned with this central
pair, showing at most one cristal plate (Fig.
21). In addition, a ring of microtubules is ob-
served surrounding the midpiece at late
stages of its development (Fig. 21). Glycogen
granules in the glycogen piece are organized
in nine tracts; one per microtubular doublet
(Fig. 24), and the transition zone between the
midpiece and the glycogen piece is marked
by a dense ring structure that is attached to
the euspermatozoan plasma membrane (Fig.
22). The latter continues to encircle the ax-
onemal microtubules, forming the end piece
of the tail (Fig. 25), without a distinct transi-
tion structure between the glycogen piece
and the end piece (Fig. 23). Cytoplasmic
bridges connect adjacent developing sper-
matids throughout various stages of eusper-
miogenesis (Figs. 3, 11).
DISCUSSION
Euspermiogenesis as seen in Clypeomorus
bifasciata and C. tuberculatus includes many
common features that were reported in all
other cerithiaceans (Giusti, 1971; Healy,
1982a, 1984; Koike, 1985; Afzelius et al.,
1989, Hodgson & Heller, 1990; Minniti 1993)
as well as other mesogastropods and neo-
gastropods (Giusti, 1969; Buckland-Nicks &
ATTIGA & AL-HAJJ
52
FIGS. 8-15.
ULTRASTRUCTURAL STUDY OF EUSPERMIOGENESIS 53
Chia, 1976; West, 1978; Griffond, 1980;
Kohnert, 1980; Healy 1983b; Koike 1985). In
general, acrosome formation is associated
with Golgi complex and involves the produc-
tion of a proacrosomal vesicle, which occurs
anywhere in the cytoplasm, because there is
no definite route of acrosome migration from
the posterior to the anterior pole of the de-
veloping spermatid. This situation was re-
ported in other cerithiaceans (Healy, 1982a,
1986a; Minniti, 1993), in contrast to many
other mesogastropods and neogastropods,
in which such a route is marked and linked to
various stages of nuclear shaping (Buckland-
Nicks & Chia, 1976; West, 1978; Jong-Brink
et al., 1977; Buckland-Nicks et al., 1983;
Jaramillo et al., 1986, Gallardo & Garrido,
1989). Cerithiids, including those used in this
study, are characterized by a high degree of
development of the pre-attachment ac-
rosome. Prior to its attachment to the nu-
cleus, the acrosome bears an acrosomal
cone, an acrosomal rod-like material and an
interstitial granule, which gives rise to the
basal plate; these constitute the elements of
the mature acrosome.
Because spermiogenesis involves several
complex processes of cellular shaping and
remodeling, microtubules and microfilaments
are expected to play a crucial rule in these
processes. In contrast to other reported cer-
ithiids, microtubules were seen within the de-
veloping acrosomal cones of Clypeomorus
bifasciata and C. tuberculatus. This was
strongly suggested by the longitudinal sec-
tions cutting through the developing ac-
rosomes (Figs. 4, 9) as well as the hollow
round structures seen in transverse sections
(Fig. 18). Such an arrangement of microtu-
bules within the cone is thought to provide it
with a degree of rigidity, and aid in its elon-
gation after it attaches to the nucleus, as was
suggested in some non-cerithiacean meso-
gastropods and neogastropods (Walker &
MacGregor, 1968; Buckland-Nicks & Chia,
1976; Giusti & Mazzini, 1973). Other cerithiids
as well as other mesogastropods and neo-
gastropods have a ring of microtubules
surrounding developing acrosomes (Buck-
land-Nicks & Chia, 1976; Huaquin & Bustos-
Obergon, 1981; Buckland-Nicks et al., 1983;
Healy, 1983b). In addition, microtubules were
seen around midpieces of Clypeomorus bi-
fasciata and C. tuberculatus at late stages of
development. Their appearance at such late
stages in these two cerithiids as well as other
mesogastropods (Jong-Brink et al., 1977;
Kohnert, 1980; Griffond, 1980; Healy, 1982a,
1983a, b, 1988b; Buckland-Nicks et al.,
1983; Afzelius et al., 1989; Al-Hajj & Attiga,
1995) strengthens the idea that they are im-
portant in sloughing the excess cytoplasm
around midpieces as well as other parts of
the euspermatozoon (Fawcett et al., 1971).
Furthermore, the ornamentation of the ma-
ture acrosomal cone with parallel plate-like
substructures seen in Clypeomorus bifasci-
ata and C. tuberculatus was also reported in
FIG. 8. C. tuberculatus. Early spermatid showing basal chromatin accumulation in nucleus (N), sites of
mitochondrial (M) association with the nucleus (arrow heads), acrosomal cone (AC), interstitial granule (IG),
and internal supporting structures (arrow) in acrosomal cone. 27,200
FIG. 9. C. tuberculatus early spermatid. Section showing acrosomal cone with microtubules (arrow) and
internal supporting structures (arrows), subacrosomal space (SAS), and interstitial granule (IG). «50,000
FIG. 10. C. bifasciata. Middle spermatid showing interstitial granule (IG), hammer-like nucleus (N), and
midpiece (MP). x22,400
FIG. 11. C. tuberculatus. Middle spermatid with a cytoplasmic bridge (asterisk) Showing acrosomal cone
(AC), external supporting structure (ES), basal plate (BP), nucleus (N), and midpiece (MP). «15,000
FIG. 12. C. tuberculatus middle spermatid. Section showing acrosomal cone (AC), internal supporting
structure (arrow heads), external supporting structure (ES), basal plate (BP), and fibrillar nucleus (N).
x44 ,000
FIG. 13. С. bifasciata middle-late spermatid. Cross section in fibrillar nucleus. «23,750
FIG. 14. C. bifasciata late spermatid. Cross section in nucleus showing islands of lamellae. x35,000
FIG. 15. C. tuberculatus late spermatid. Cross section in nucleus with semi-fully condensed chromatin.
х31,500
ATTIGA & AL-HAJJ
54
oe
A A À
RO = A At RU
nn TE NS D
A
Я
es us si,
LT, 53. ATA +
nn in
Ly
Pr
FIGS. 16-25.
ULTRASTRUCTURAL STUDY OF EUSPERMIOGENESIS 55
other mesogastropods and neogastropods
(Giusti & Mazzini, 1973; Healy, 1983a, 1986b;
Jaramillo et al., 1986; Afzelius et al., 1989;
Al-Hajj & Attiga, 1995). Acrosomal mem-
branes in Chorus giganteus (Jaramillo et al.,
1986), Truncatella subcylindrica (Giusti &
Mazzini, 1973), and Melanopsis (Afzelius et
al., 1989) were reported to have a scalloped
appearance with regular periodicity. This or-
namentation of the growing acrosome that is
later hidden by electron-dense material
seems to be of scarce occurrence. Giusti &
Mazzini (1973) interpreted this periodicity as
microtubular palisade, whereas Jaramillo et
al. (1986) thought that it is due to the pres-
ence of actin crests, which may play a role in
acrosome reaction and egg penetration.
However, the lack of Knowledge about ac-
rosome reaction and fertilization in gastro-
pods makes it difficult to conclude the nature
or function of these structures, pending fur-
ther investigation.
Chromatin condensation and nuclear shap-
ing are two highly linked processes in sper-
miogenesis of mesogastropods and neo-
gastropods. Chromatin condensation passes
through granular, fibrillar and lamellar phases,
culminating in a homogeneous compact nu-
cleus with no ultrastructure (Walker & Mac-
Gregor, 1968; Buckland-Nicks & Chia, 1976;
Feral, 1977; West, 1978; Huaquin & Bustos-
Obergon, 1981; Healy, 1982a, b, 1983b,
1988b; Buckland-Nicks et al., 1983; Jaramillo
et al., 1986; Gallardo & Garrido, 1989). The
mature nucleus in the two cerithiids investi-
gated in this study has a short basal invagi-
nation accommodating the proximal portion
of the axoneme, which is a common nuclear
shape seen in many other mesogastropods
and neogastropods (Giusti, 1969, 1971; Giusti
& Mazzini, 1973; Reader, 1973, Griffond,
1980; Kohnert, 1980; Koike & Nishiwaki, 1980;
Healy, 1982a, b, 1983a, 1986b). On the other
hand, some mesogastropods and many neo-
gastropods have intranuclear canals that in-
vaginate the nucleus completely up to its apex
(Walker & MacGregor, 1968; Buckland-Nicks,
1973; Buckland-Nicks & Chia, 1976; West,
FIG. 16. С. tuberculatus mature euspermatozoon, with acrosome (A) and nucleus (N). x48,000 Inset:
Section showing acrosomal cone (AC) with plate-like substructure (arrow head), acrosomal rod-like material
(AR), and basal plate (BP). x57,500
FIG. 17. C. bifasciata semi-mature acrosome. Section showing acrosomal cone (AC) with plate-like sub-
structure (arrow head), acrosomal rod-like material (AR), and basal plate (BP). x63,000
FIG. 18. C. bifasciata semi-mature euspermatozoon. Section in acrosome showing acrosomal rod-like
material (AR), microtubules (MT) in acrosomal cone, and surrounding pseudopodium (PP) of nutritive cell.
x40,000
FIG. 19. C. tuberculatus semi-mature euspermatozoa showing nucleus (N), centriolar derivative (CD) in
nuclear basal invagination and midpiece with mitochondrial sheath (M) surrounding the axoneme (AX).
x60,000
FIG. 20. C. tuberculatus semi-mature euspermatozoon. Cross section in nucleus (N) showing the proximal
portion of the axoneme (arrows) within nuclear basal invagination. Notice pseudopodia (PP) of nutritive
cells. x40,000
FIG. 21. С. bifasciata late euspermatozoon. Cross section in midpiece showing two large and two extremely
small mitochondrial (M) elements surrounded by microtubules (arrows). x48,000
FIG. 22. C. tuberculatus mature euspermatozoon. Section showing dense ring structure (DRS) at the
junction between midpiece with mitochondrial sheath (M) around axoneme (AX), and glycogen piece (GP).
x38,000
FIG. 23. C. bifasciata mature euspermatozoa. Sections showing the junction between glycogen piece (GP)
and end piece (EP). x52,000
FIG. 24. C. bifasciata mature euspermatozoa. Cross section in glycogen piece showing nine tracts of
glycogen granules associated with axonemal microtubular doublets. x37,500
FIG. 25. С. bifasciata mature euspermatozoa. Cross section in end piece showing 9 + 2 pattern of micro-
tubular arrangement surrounded by plasma membrane. x128,000
56 ATTIGA 8 AL-HAJJ
1978; Huaquin & Bustos-Obergon, 1981;
Buckland-Nicks et al, 1983; Healy, 1984;
Jaramillo et al., 1986; Hodgson, 1993). Healy
(1983a) interpreted this extreme structural di-
versity in the extent of nuclear invagination to
factors in the reproductive environment, es-
pecially because some superfamilies include
both types of nuclei among their species
(Buckland-Nicks, 1973; Healy, 1988a).
Centrioles were not seen in developing eu-
spermatids in C. bifasciata and C. tubercula-
tus, and the proximal portion of the axoneme
was attached to the posterior nuclear invagi-
nation through a single dense structure that
has no apparent microtubules. This centriolar
derivative was reported in other caeno-
gastropods (Buckland-Nicks, 1973; Healy,
1982a, 1983a, 1986a, b, 1988b).
The midpiece in euspermatozoa of C. bi-
fasciata and C. tuberculatus is consistent with
those of other cerithiids and members of sub-
group 1(i) of Healy’s (1983a) classification of
mesogastropods as a group of caenogastro-
pods. Midpieces in these animals are char-
acterized by four non-helically arranged ele-
ments, two of which are extremely small,
whereas the other two are large showing mul-
tiple cristal plates. The non-helical arrange-
ment of mitochondria around the axoneme in
Cerithiacea is considered a primitive charac-
ter compared to the helical mitochondrial
sheath seen in other mesogastropods and
neogastropods (Walker & MacGregor, 1968;
Giusti, 1969, 1971; Anderson & Personne,
1970; Buckland-Nicks, 1973; Giusti & Maz-
zini, 1973; West, 1978; Koike & Nishiwaki,
1980; Kohnert, 1980; Griffond, 1980). This
feature supports Healy's (1982a) proposi-
tion that cerithiaceans represent ancestral
mesogastropods, acting as a linkage group
between primitive spermatozoa of Archaeo-
gastropoda on one hand and modified sper-
matozoa of higher mesogastropods and neo-
gastropods on the other. Such a position
makes it an interesting group to study sper-
matozoan evolution.
In cerithiids, including those investigated in
this study, the glycogen piece consists of
nine tracts of glycogen granules associated
with the nine doublets of the axonemal mi-
crotubules. This arrangement, which is seen
also in other mesogastropods and neogas-
tropods (Giusti, 1969, 1971; Buckland-Nicks,
1973; Reader, 1973; West, 1978; Koike &
Nishiwaki, 1980; Kohnert, 1980; Huaquin &
Bustos-Obergon, 1981; Healy, 1982a, 1986a,
b, 1988a, c; Jaramillo et al., 1986; Gallardo 8
Garrido, 1989; Al-Hajj & Attiga, 1995), is
thought to be linked to the euspermatozoan
motility, because the nine glycogen tracts in
the mature euspermatozoa obscures nine ra-
dial links between the axonemal doublets
and the plasma membrane in the immature
spermatid (Healy, 1983a).
Many investigators have suggested that
acrosomal ultrastructure of gastropods could
provide useful taxonomic data by being spe-
cies-specific. In Cerithiacea, such a species-
specific acrosome ultrastructure is not well
presented. Species-specific features were
seen when comparing acrosomes of Cerith-
¡um vulgatum (Giusti, 1971), C. nodulosum
(Koike, 1985), C. rupestre (Minniti, 1993), C.
caeruleum (Al-Hajj & Attiga, 1995), whereas
Clypeomorus bifasciata, C. tuberculatus (this
study), C. moniliferus (bifasciata), and C.
breviculus (Healy, 1983a) have almost identi-
cal acrosomes. In addition, differences in the
ultrastructure of the acrosome were estab-
lished at the generic level between Cerithium,
Rhinoclavis, Australaba (Healy, 1983a), and
Clypeomorus (Healy, 1983a; this study), but
were not seen between euspermatozoa of
Conomurex and Lambis, which possess very
similar acrosomes (Koike & Nishiwaki, 1980).
In conclusion, comparative studies of
sperm ultrastructure have proved to act as an
acceptable guide for determining the affini-
ties between major groups in gastropods as
well as in many other animal groups.
ACKNOWLEDGMENTS
The authors are thankful to Dr. Saleem Al-
Mograby from the Marine Science Station of
the Gulf of Aqaba for help in providing access
to collect the specimens from the station’s
rocky beach.
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Revised Ms. accepted 28 November 1995
MALACOLOGIA, 1996, 38(1-2): 59-65
CA REGULATION IN THE FRESHWATER BIVALVE ANODONTA IMBECILIS:
I. EFFECT OF ENVIRONMENTAL CA CONCENTRATION AND BODY MASS ON
UNIDIRECTIONAL AND NET CA FLUXES
Dazhong Xu' & Michele С. Wheatly'
Department of Zoology, University of Florida, Gainesville, Florida 32611, USA
ABSTRACT
The present paper reports unidirectional and net Ca fluxes of a freshwater bivalve, Anodonta
imbecilis, as a function of external Ca concentration and body mass. Larger animals were better
able to maintain Ca balance than smaller animals, which experienced net loss of Ca. External
Ca concentration had no significant effect on net Ca flux. Unidirectional Ca influx decreased
with body mass and increased with external Ca concentration. The relationship between ex-
ternal Ca concentration and unidirectional Ca influx follows the Michaelis-Menten equation. The
estimated half saturation Ca concentration for unidirectional Ca influx and the maximum uni-
directional influx were 0.213 mM and 4.329 ито! g dry mass 'h ', respectively. External Са
concentration did not affect unidirectional Ca efflux of the animals. Unidirectional Ca efflux
decreased with body mass.
Key words: Calcium flux, calcium concentration, body mass, bivalve.
INTRODUCTION
While calcification has been relatively well
studied in molluscs (reviewed by Watabe,
1983), the contribution made by whole body
unidirectional Ca flux to Ca regulation in bi-
valves has not been well established.
In molluscs, various epithelia can take up
external Ca (Van der Borght, 1963; Van der
Borght & Van Puymbroeck, 1964; Green-
away, 1971; Thomas et al., 1974), especially
the mantle surface facing the mantle cavity
(Jodrey, 1953; Horiguchi, 1958) and the gill
(Horiguchi, 1958). The active transport of Ca
was demonstrated in the freshwater gastro-
pods Lymnaea stagnalis (Van der Borght &
Van Puymbroeck, 1964; Greenaway, 1971)
and Biomphalaria glabrata (Thomas et al.,
1974). Based on models for active Ca uptake
into other freshwater species (for example te-
leost fish, Flik et al., 1985), the epithelial up-
take occurs in two stages: diffusion down an
electrochemical gradient across the apical
membrane into the cytosol, and active trans-
port across the basolateral membrane into
the haemolymph. If, as in other freshwater
species, the Ca pump in molluscan epithelia
is Ca-activated ATPase (Watabe, 1983), the
rate of Ca uptake should be correlated with
the Ca concentration in the ambient water.
Previous studies showed that this is true for
some molluscs (Greenaway, 1971; Thomas
et al., 1974; Russell-Hunter, 1978; Pynnonen,
1991) but not others (Hunter, 1975; Russell-
Hunter et al., 1981).
Allometry refers to the scaling of physio-
logical function/morphological parameters to
body mass. Interspecific allometry of captive
aquatic molluscs described scaling of water
flux to body mass (Nagy & Peterson, 1988).
Significant relationships between dry body
mass and shell length, shell height, gut-pas-
sage time, gut content or metabolic fecal loss
have also been reported (Hawkins et al.,
1990). The relationship between dry mass
and oxygen consumption had a negative cor-
relation (Dietz, 1974).
The present study uses radiotracer tech-
niques to determine the unidirectional Ca
fluxes in a freshwater bivalve, Anodonta im-
becilis, as a function of external Ca concen-
tration and body mass.
MATERIALS AND METHODS
Experimental Animals and General
Holding Conditions
The freshwater bivalves Anodonta imbeci-
lis (6-58 g) were collected 60 km from
Gainesville, Florida, from a canal along the
‘Present Address: Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA.
59
60 XU & WHEATLY
TABLE 1. Wet and dry mass of animals used to determine the effect of body mass on unidirectional
and net Ca flux.
group 1 group 2 group 3 group 4
wet mass (g) 15 == 0:89 15.88 + 0.56 25.98 + 1.40 44.02 + 2.03
dry mass (9) 0.362 + 0.041 0.511 + 0.064 1.061 + 0.076 1.929 + 0.171
Mean + SEM. М (groups 2, 3, 4) = 10. М (group 1) = 9.
Suwannee River at Fanning Springs. The ап-
imals collected were kept in aquaria with
aged and well-aerated 21°C Gainesville tap
water with the following cationic composition
(in mM): Nat, 0.55; К*, 0.04; Са?*, 0.60;
Mg?*, 0.42; and СГ, 0.73. The pH was 7.7.
Food was withheld for the holding period (up
to two months), and animals were used
within two months of collection. Animals
were acclimated in aquaria for at least 10
days before measurements were made. Only
healthy animals (indicated by relatively heavy
weight, active ventilation and powerful water
ejection upon disturbance) were used in the
experiments. All experiments were con-
ducted at 21°C.
Unidirectional and Net Ca Fluxes—Effect of
External Ca Concentration
Four groups of animals were used in the
experiment. The Ca concentrations of the ex-
perimental media were 0.27, 0.60, 1.00 and
2.00 mM, respectively. The outer surface of
the shell of the experimental animals was
covered with wax to prevent direct Ca loss
from the shell/water interface. Animals were
acclimated in the experimental water for 3
days before conducting the experiment. Me-
dia with Ca concentration of 1.00 and 2.00
mM were made by adding CaCl. to Gaines-
ville tap water (0.6 mM Ca). The medium with
Ca concentration of 0.27 mM was made us-
ing the following recipe (in mM): NaCl, 0.4;
CaCl,, 0.27; NaHCO,, 0.2; and KCI, 0.04. An-
imals were placed individually in experimen-
tal flux chambers containing 300 ml medium
and acclimated for more than 12 hours. At
the beginning of a flux measurement, the wa-
ter was drained from the chamber and 200 ml
fresh medium were added. An initial water
sample was taken from each chamber and
then 1 uCi of *°Ca (CaCl, in water, 10 mCi
ml *, Du Pont) was added to each chamber.
Water samples were taken from each cham-
ber at t= 0h and t = 6 В for determination of
radioactivity and Ca concentration. These
samples were used to estimate net and uni-
directional Ca fluxes. At the end of the exper-
iment, animals were sacrificed by cutting the
adductor muscles using a dissecting knife.
Soft tissues of each animal were then dis-
sected out and dried to constant weight to
determine dry mass. In a parallel experiment,
empty shells (the valves sealed together and
covered with wax on the outer surface) were
bathed in an identical chamber to estimate
the possible accumulation of *°Ca by the
shell surface. Throughout the paper, dry
mass means the dried mass of the soft tis-
sues (excluding shells), wet mass refers to
the whole wet mass of the animals (including
shells).
Unidirectional and Net Ca Flux—Effect of
Body Mass
Bivalves with wet mass of 6-58 g (N = 39)
and dry mass of 0.2-2.7 g were used in the
experiment. Animals were divided into four
groups according to wet mass (Table 1).
Groups were numbered 1 to 4 (small to
large). For each group, the flux volume and
isotope addition were as follows: group 1,
100 ml and 0.5 uCi *°Ca; group 2 and 3, 150
ml and 0.8 uCi “Ca; group 4, 200 ml and 1
uCi *°Ca. The experimental method was the
same as described above. The Ca concen-
tration of the media was 1 mM.
Analytical Methods
Water samples (3 ml) were mixed with
ScintiVerse fluor (3 ml) and then radioactivity
was measured using a liquid scintillation
counter (LSC, Beckman LS5801). The Ca
concentration of experimental water or extra-
pallial fluid (EPF) was measured after appro-
priate dilution (0.2 ml sample + 2 ml 2% LaCl,
+ 1.8 ml distilled water) using an atomic ab-
sorption spectrophotometer (Perkin Elmer
2100).
Calculation
The flux equation described by Wheatly
(1989) was used to calculate unidirectional
Ca influx:
Ca REGULATION IN THE FRESHWATER BIVALVE ANODONTA IMBECILIS 61
Jin = =——— (1)
where Jin is unidirectional Ca influx (umol g
dry mass 'h '), Ri and Rf are the initial and
final radioactivity (cpm ml ') of respective
water samples, V is the flux volume (ml), SA is
the medium mean specific radioactivity (cpm
umol ') calculated as the mean radioactivity
divided by the mean Ca concentration, t is
the elapsed time (h), and W is the dry mass of
the animal (9).
Net flux was calculated as:
(EL—IEHV
Hehe? (2)
tW
where Jnet is Ca net flux (umol g dry mass
h '), Ci and Cf are the initial and final me-
dium Ca concentrations (mM), V is the flux
volume (ml), t is the elapsed time (h), and W is
the dry mass of the animal (g). A positive
value for net flux indicates net Ca influx while
a negative value indicates net Ca efflux.
Unidirectional efflux was calculated using
the conservation equation:
1
Jout = Jin — Jnet (3)
Statistical Analysis
Data were expressed as mean and stan-
dard error (+ SEM). The statistical analysis
was performed using StatView 4.01 and Su-
per ANOVA computer packages. Correlation
analysis was performed by calculating corre-
lation coefficients (r values) and using Fish-
er's rto z method to test the significance of
correlation. One-factor ANOVA was used to
analyze differences between groups and
then Fisher's PLSD test was used when nec-
essary to compare the means. ANCOVA was
used to compare the slopes and intercepts of
different linear relationships. The significance
level for all statistical analyses was set at
0.05.
RESULTS
Empty shells showed no significant accu-
mulation of “Са on the waxed outer shell
surface. Ca net flux was affected by the Ca
concentration in the medium (Fig. 1). Animals
in medium containing 0.27 mM Ca showed a
significant net Ca efflux of — 1.63 + 0.24 umol
g dry mass 'h ' (М = 9) compared to those
D Ori © M NO vw» y
Ca flux (umol g dry mass hl
do
A
0 0.5 1 1.9 2
Ca concentration in the medium (mmol I!)
FIG. 1. Unidirectional and net fluxes of Anodonta
imbecilis in media of different Ca concentrations.
Points represent mean and standard errors. The
wet mass of animals used were as follows: group 1
(Ca = 0.27 mM), 41.59 + 2.30 g (N = 9); group 2 (Ca
= 0.6 mM), 40.14 + 2.41 g (N = 10); group 3 (Ca =
1.0 mM), 44.02 + 2.03 g (N = 10); group 4 (Ca = 2.0
mM), 43.59 + 2.07 g (N = 8). The equation of the
curve fitting the influx data is: influx 4.329C/(0.213
+ C), where C is the Ca concentration of the me-
dium; r = 0.996.
of the animals in media containing 0.60 mM
(Fisher’s PLSD, p = 0.0036), 1.00 mM (Fish-
er's PLSD, p = 0.0007) and 2.00 mM Ca
(Fisher’s PLSD, p = 0.0001). There were no
significant differences in Ca net flux in Ca
concentrations of 0.60, 1.00, 2.00 mM (Fish-
er’s PLSD, p = 0.1500 for the largest differ-
ence).
There was a nonlinear relationship be-
tween the unidirectional Ca influx and the ex-
ternal Ca concentration (Fig. 1). The unidirec-
tional influxes for animals in media containing
0.27, 0.60, 1.00 and 2.00 mM Ca were sig-
nificantly different (one-factor ANOVA, p =
0.0132). The mean unidirectional influx in-
creased as external Ca concentration in-
creased, was partially saturable and could be
approximately described by the Michaelis-
Menten equation:
Influx = К (4)
m+C
where K is the maximum rate of unidirec-
tional Ca influx, Km is the Ca concentration in
the medium at which half saturation is at-
tained, and C is the Ca concentration in the
62 XU & WHEATLY
dry mass = 0.047(wet mass) -0.184 e
Dry mass (g)
Wet mass (g)
FIG. 2. Relationship between dry mass and wet mass of Anodonta imbecilis. Points represent the mass of
individual animals; r = 0.909 for regression. Fisher’s r to z, p < 0.0001, N = 39.
medium. The calculated half saturation Ca
concentration for unidirectional Ca_ influx
(Km) was 0.213 mM. The maximum unidirec-
tional influx (K) was 4.329 umol g dry mass
'h '. Thus, the following equation can be
used to describe the relationship between
unidirectional influx and external Ca concen-
tration:
C
ntux=4:329 == (5)
0.213+C
Unidirectional Ca effluxes showed no sig-
nificant difference among animals in media
with different Ca concentration (one factor
ANOVA, p = 0.7807; Fig. 1).
The dry mass of bivalves (excluding shell)
was positively correlated with wet mass (r =
0.909, Fisher's r to z, p < 0.0001, Fig. 2).
Larger animals generally maintained Ca bal-
ance as indicated by the negligible net flux
(Fig. 3). However, animals smaller than 0.5 g
tended to exhibit a negative Ca balance as
indicated by a net efflux (Fig. 3). Both the
unidirectional Ca influx and efflux decreased
with increase in dry body mass. Negative lin-
ear relationships were derived between log
unidirectional Ca influx and log dry mass (r =
— 0.800, Fisher's r to z, p < 0.0001), and log
unidirectional efflux and log dry mass (r =
— 0.862, Fisher's r to z, p < 0.0001; Fig. 4).
The slopes and intercepts respectively were
as follows: —4.03, 6.52 (unidirectional influx)
and —8.34, 7.54 (unidirectional efflux). The
slope and intercept for unidirectional efflux
were both significantly higher than those for
unidirectional influx (ANCOVA, p = 0.0001).
DISCUSSION
Active unidirectional Ca influx that follows
enzyme saturation kinetics has been previ-
ously demonstrated in freshwater snails.
Greenaway (1971) found in the snail Lymnaea
stagnalis that active uptake of Ca was nec-
essary below external levels of 0.5 mM. The
uptake mechanism was half-saturated and
near-saturated in external media containing
0.3 and 1.0-1.5 mM Ca, respectively, and
snails showed a positive Ca balance in media
containing more than 0.062 mM Ca. For the
snail Biomphalaria glabrata, the half and near
saturated Ca concentration for Ca uptake
were 0.267 and 1.0-2.0 mM, respectively,
and the minimum equilibrium concentrations
were 0.012-0.025 mM for a closed system
and 0.25 mM for an open system (Thomas et
al., 1974). Both animals exhibited a high af-
finity Ca uptake mechanism. In the present
study, the unidirectional Ca influx of A. imbe-
cilis seems to display the same kinetics as a
function of external Ca concentration. The
half saturation Ca concentration in the me-
dium was 0.213 mM, lower than the value
estimated for the freshwater snail L. stagnalis
(0.3 mM; Greenaway, 1971) and the value es-
Ca REGULATION IN THE FRESHWATER BIVALVE ANODONTA IMBECILIS 63
m 5
я 0
a
3 SN
a = -10
FE
a -15
O E -20
3 -25
do
=
0 р
Dry mass (2)
FIG. 3. Relationship between Ca net flux and body dry mass of Anodonta imbecilis. Points represent Ca net
flux of individual animals. The Ca concentration of the medium is 1 mM. N = 36.
—
o
o
Ca flux (umol g dry mass Ih’)
0.1
log (influx) = -0.4028log (dry mass) + 0.6520
log (efflux) = -0.8342log (dry mass) + 0.7548
—e—influx
+ efflux
Dry mass (g)
FIG. 4. Relationship between log unidirectional Ca influx and log dry mass (r = 0.800, Fisher's r to z, p <
0.0001), and between log unidirectional Ca efflux and log dry mass (r = 0.862, Fisher's r to z, p < 0.0001)
of Anodonta imbecilis. Points represent the unidirectional influx or efflux of individual animals. The slopes
for log unidirectional influx and log unidirectional efflux are significantly different (ANCOVA, p = 0.0001). N
= 36.
timated for the freshwater snail Biomphalaria
glabrata (0.267 mM; Thomas et al., 1974).
If the unidirectional Ca efflux is purely by
passive diffusion, one would expect unidirec-
tional efflux to decrease as the external con-
centration is raised. Because unidirectional
Ca efflux was unaffected by change in exter-
nal Ca concentration, other mechanisms may
be involved. Greenaway (1971) suggested
that part of the unidirectional Ca influx in L.
stagnalis is due to exchange diffusion and
that this component increases when external
Ca concentration increases following en-
zyme-saturation kinetics. If the unidirectional
64 XU & WHEATLY
Ca efflux in A. imbecilis is attributable to ex-
change diffusion, then unidirectional efflux
would increase as external Ca rises. Any por-
tion of unidirectional Ca efflux not attributed
to exchange diffusion (‘routine loss””) would
decrease with the increase in external Ca
concentration because of the reduction in
concentration gradient. The combined effect
might be that unidirectional Ca efflux is un-
changed. The net Ca flux of animals in media
of different Ca concentration mirrored the
change in unidirectional Ca influx because
unidirectional Ca efflux remained constant.
This pattern of Ca net flux is similar to the Ca
net uptake pattern of L. stagnalis in media of
different Ca concentration (Greenaway,
1971). The difference between these two an-
imals is that in A. imbecilis Ca net flux was
negative as opposed to the net uptake exhib-
ited by L. stagnalis.
The relationship between wet and dry
mass of A. imbecilis is linear, indicating the
proportional increase of soft body tissue with
shell and water content. A similar relationship
was found between shell weight and fresh
tissue weight in the freshwater snail L. stag-
nalis (Greenaway, 1971).
Few animals in this study exhibited a sig-
nificant net uptake of Ca from the medium,
suggesting that considerable Ca is obtained
from dietary sources. The freshwater snail
Lymnaea stagnalis was found to obtain 20%
of its calcium from food (Van der Borght &
Van Puymbroeck, 1966). This is also consis-
tent with previous work, which demonstrated
that freshwater bivalves obtain part of their
Ca from food (Pynnonen, 1991).
Larger animals are better able to maintain
their Ca balance with the environment than
smaller animals, which tend to lose Ca to the
medium. This was due to the fact that unidi-
rectional Ca efflux is larger than Ca influx in
small animals and decreases at a greater rate
with increase in body mass. This implies that
smaller animals depend more on dietary Ca
than larger animals, possibly since they cal-
cify their shell more rapidly.
Allometric regression showed that unidi-
rectional Ca fluxes decreased with dry body
mass of A. imbecilis. Thus, smaller animals
exchange Ca with their environment more
rapidly commensurate with an increased sur-
face area to volume ratio. This result is similar
to a recent study on crayfish, which revealed
that the diffusional and active ¡on flux rates
are both greater in small crayfish (Wheatly et
al., 1991). An allometric study of Na fluxes in
amphibia (Pruett et al., 1991) showed that re-
gression lines for unidirectional Na influx and
efflux had the same slope and intercept con-
firming Na balance in animals of all size. In
the present study, efflux decreased more
than influx with increase of body dry mass
resulting in significantly greater net efflux of
Ca in smaller animals.
ACKNOWLEDGMENTS
This research was supported by NSF grant
DCB 89 16412 to MGW. We thank Drs. Karen
Bjorndal, David Evans and Frank Nordlie for
their suggestions on the research and for use
of experimental equipment. We thank Dr. Jim
Williams, Ms. Jane Brimbox and Mr. Ricardo
Lattimore in the U.S. Fish and Wildlife service
for their help in collecting experimental ani-
mals.
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Revised Ms. accepted 1 January 1996
MALACOLOGIA, 1996, 38(1-2): 67-85
MICROSCULPTURES OF CONVERGENT AND DIVERGENT POLYGYRID
LAND-SNAIL SHELLS
Kenneth C. Emberton
Department of Malacology, Academy of Natural Sciences of Philadelphia, 1900 Benjamin
Franklin Parkway, Philadelphia, Pennsylvania 19103-1195, U.S.A.
ABSTRACT
Polygyrid evolution has produced five pairs of closely convergent shell forms, four of which
occur in sympatry. Scanning electron microscopy of the apertural parietal and basal denticles
(or regions) (at about 500x) in those ten species, and of the body whorl (at about 100x) in those
and eleven more polygyrid species, reveals possible new microsculptural characters, homol-
ogies, and radiations. Twelve informative new character states are tentatively proposed, of
which half support, without homoplasy, previous shell-free phylogenetic hypotheses based on
anatomy and allozymes. Two of the homoplastic characters actually enhance shell-form con-
vergences, which are nonetheless distinguishable using other microsculptural features. Further
SEM studies are warranted to test these proposed characters, to add others, and to test the
hypothesis that shell micromorphology is much more informative than shell macromorphology
for land-snail phylogenetics.
Key words: Gastropoda: Stylommatophora, morphology, systematics, phylogenetics, cla-
distics, SEM.
INTRODUCTION
Polygyrid shell-form evolution is unique for
its multiple close convergences in sympatry
and is also noteworthy for its sudden diver-
gences; shell-sculpture evolution in polygy-
rids is also of great interest for its repeated
convergences on periostracal hairs and its
divergences among sister taxa (Pilsbry, 1940;
Solem, 1976; Emberton, 1988, 1991a, 1994a,
1995a, 1995b; Asami, 1988, 1993). These
combinations of close convergences and
rapid divergences make it virtually impossi-
ble to reconstruct polygyrid phylogeny from
gross shell morphology, even when develop-
mental characters are viewed by x-ray (Em-
berton, 1995b). Polygyrid shell convergences
and divergences, however, have so far been
compared only macroscopically, at magnifi-
cations no greater than 50x.
The purpose of this paper is a preliminary
assessment of the the microsculptures of se-
lected polygyrid shell convergences and di-
vergences, using scanning electron micros-
copy (SEM).
Four species of polygyrids have previously
been examined under SEM for microsculp-
tural features of the apertural lip: Stenotrema
barbatum (Clapp) (Solem, 1972: figs. 23, 24
= Solem, 1974: fig. 5), as well as Daedalochila
67
auriformis (Bland), Millerelix mooreana (Bin-
ney), and M. doerfeuilliana sampsoni (Weth-
erby) (Solem & Lebryck, 1976: figs. 33-46).
All four species had fields of hexagonal to
rounded crystalline plates, uplifted on one
side, and those plates varied in size and dis-
tributional pattern among species. Intraspe-
cific variation was studied in D. auriformis,
with the important discoveries that the pari-
etal and the palatal apertural denticles dif-
fered in microsculpture, and that a gerontic
shell had more strongly developed micro-
sculpture than a younger adult shell (Solem &
Lebryck, 1976).
Only a single polygyrid specimen has pre-
viously been examined under SEM for shell
periostracal microsculpture. Stenotrema bar-
batum exhibited at 195x and 360x a regular
array of gradually tapered, sharp-pointed
hairs, seemingly round in cross-section and
projecting perpendicularly from a surface
field of subparallel, slightly anastomosing
ridges (Solem, 1974: fig. 6).
The present study is a preliminary survey,
based on only one shell per species (al-
though including several pairs of sister spe-
cies), so the microsculptural characters dis-
covered herein must be considered tentative.
In order to minimize the known sources of
intraspecific variation (Solem & Lebryck,
68 EMBERTON
1976), only gerontic shells were used and
both parietal and palatal apertural denticles
(or regions) were examined.
MATERIALS AND METHODS
Twenty-one polygyrid species were cho-
sen for examination; Figure 1 presents their
phylogenetic relationships as hypothesized
from anatomical and biochemical data (Em-
berton, 1988, 1991a, 1994a, 1995b). The
species include North America's four most
extreme cases of shell-form convergence in
sympatry (Emberton, 1995b: fig. 1): globose
Neohelix major and Mesodon normalis, um-
bilicate Allogona profunda and Appalachina
sayana, flat Xolotrema fosteri and Patera lae-
vior, and tridentate Triodopsis fallax and In-
flectarius inflectus. A fifth shell-form conver-
gence (Emberton, 1991b) was also included:
“lipped” Neohelix dentifera and Inflectarius
ferrissi.
Additional polygyrid species were included
for their periostracal-microsculpture diver-
gences and convergences. Xolotrema deno-
tata and X. obstricta are sister species (Em-
berton, 1988) that can hybridize in the field
(Vagvolgyi, 1968) and in the laboratory
(Webb, 1980), but their differences in shell-
whorl shape and sculpture are extreme. Xo-
lotrema obstricta has a strongly keeled pe-
riphery and is sculpted with large, strongly
raised ribs, whereas X. denotata has a
rounded periphery and is sculpted with hair-
like processes (Pilsbry, 1940; Emberton,
1988). The keeled, ribbed shell of X. obstricta
is closely paralleled by that of Patera sargen-
tiana (Pilsbry, 1940; Emberton, 1991a), with
which it is sympatric in northern Alabama.
Species of the Patera radiation (Emberton,
1991a) have diverged primarily in their shell
surface sculpture: P. laevior is smooth, P.
sargentiana is ribbed, P. perigrapta bears in-
cised spiral grooves, and P. appressa sculp-
tior is pustulose (Pilsbry, 1940).
Hair-like periostracal processes on the
shell surface have arisen independently and
convergently (Emberton, 1995b) in Xolotrema
denotata, in some Vespericola such as V. co-
lumbiana pilosa, in the Stenotrema clade,
and in the /nflectarius clade (Pilsbry, 1940).
Stenotrema's radiation is marked by extreme
divergence in shell hairs, the variation of
which includes short and dense (e.g. S. max-
illatum), and long and sparsely distributed
(e.g. S. barbigerum) (Pilsbry, 1940). To a
much smaller degree, the general shapes of
shell hairs also seem (at 50x) to differ among
species of /nflectarius: broad-based and
sharp-tipped in I. inflectarius and I. magazin-
ensis, acutely triangular in /. smithi, obtusely
triangular in /. subpalliatus, and lost in I. fer-
rissi, the sister species of I. subpalliatus (Em-
berton, 1991a).
All studied shells are in the collection ofthe
Academy of Natural Sciences of Philadelphia
(ANSP). Species authors and ANSP catalog
numbers of vouchers are given in the figure
captions.
Shells were prepared for SEM using meth-
ods modified slightly from Solem (1970):
soaking overnight in a weak solution of de-
tergent (Alconox), immersing for five to 20
seconds in an ultrasonic cleaner, rinsing in
distilled water, air-drying, and mounting—in
standard position—on stubs using some
combination (depending on the size of the
shell) of double-sided conductive tape, car-
bon paint, carbon cement, carbon paste, and
custom-bent paper clips. Mounted shells
were gold-coated and photographed with a
Cambridge Stereoscan 200 SEM in one or
more of the following views: (a) whole shell
(or as much as possible, including the entire
aperture) in apertural view; (b) edge of pari-
etal denticle (or callus) at about 1,000x; (c)
edge of basal denticle (or lamellum or lip) at
about 1,000x; and (d) body-whorl sculpture
at about 200x and at <100x if necessary.
The resulting photographs were descrip-
tively compared, then subjected to a stan-
dard phylogenetic character analysis (Wiley,
1981; Brooks & McLennan, 1991: chapter 2).
The states of each character were parsimo-
niousiy mapped by hand on the polygyrid
phylogenetic hypothesis (Emberton, 1988a,
1991a, 1994a, 1995b).
RESULTS
Four Convergences in Sympatry
Figures 2-5 show SEM photographs of the
four pairs of convergent species that occur in
sympatry, with triodopsins and Allogona on
the left, and mesodontins on the right: a, b,
Neohelix major and Mesodon normalis; c, d,
Xolotrema fosteri and Patera laevior; e, f, Tri-
odopsis fallax and Inflectarius inflectus; and
g, h, Allogona profunda and Appalachina say-
ana. Apertural views at lowest possible mag-
nifications (Fig. 2) indicate extremely close
convergences in apertural dentition between
POLYGYRID SHELL MICROSCULPTURE 69
= Neohelix major 2a-5a
[== — Г
| С tus N. dentifera 6a,c,e,g
L
Kal
| | Pr Xolotrema fosteri 2c-5c
| Si
| Fail FF X. denotata 7a,c,e
—— Load |
| | == X. obstricta 7b,d,f
| 108! |
| LL Triodopsis fallax 2e-5e
=— li
| ААА Vespericola columbiana Эа
U
mt |
| | [=== Stenotrema maxillatum 9c
== | EL Stenotrema barbigerum 9e, 10d
=== Patera perigrapta 8c
=== === P. laevior 24-54
| | === P, appressa 8e
| LE P. sargentiana 8a,b,d
= Inflectarius subpalliatus Of
| zz I. ferrissi 6b,d,f,h,10a,c
| IT | == /. magazinensis 9b
| == |, smithi 9d
== /, inflectus 2f-5f,10b
| ra Appalachina sayana 2h-5h
цы Mesodon normalis 2b-5b
FIG. 1. Phylogenetic relationships of the 21 species examined for this study, as hypothesized from alloz-
ymes and reproductive morphology and behavior (Emberton, 1988, 1991a, 1994a). Principal convergences
are designated by five abbreviations for shell shape/size: G, globose; F, flat; T, tridentate; U, umbilicate; L,
lipped (Emberton, 1991b, 1995b). Figure numbers of SEM photos are given for each species name;
“2a-5a” = 2a, 3a, 4a, 5a.
N. major and M. normalis (without dentition) fallax and I. inflectus (parietal denticles similar
and between X. fosteri and P. laevior (blade- but more curved in T. fallax, palatal denticles
like parietal denticles and basal lamellae); nearly identical, basal denticles similar but
moderately close convergence between T. with a columellar buttress in T. fallax); and
70 EMBERTON
FIG. 2. Apertural features of North America's four most extreme cases of polygyrid shell-form convergence
in sympatry (Emberton, 1995b: fig. 1). A, Neohelix major (Binney), ANSP uncataloged, 5.65x. B, Mesodon
normalis (Pilsbry), ANSP uncataloged, 5.50x. C, Xolotrema fosteri (F. C. Baker), ANSP 117483, 4.43x. D,
Patera laevior (Pilsbry), ANSP 186465, 3.98x. E, Triodopsis fallax (Say), ANSP 192768, 4.95x. F, Inflectarius
inflectus (Say), ANSP 91616, 6.30x. G, Allogona profunda (Say), ANSP 77867, 4.90x. H, Appalachina
sayana (Pilsbry), ANSP 264654, 5.35x.
POLYGYRID SHELL MICROSCULPTURE 71
FIG. 3. The parietal denticles or parietal regions of the same specimens as Fig. 2, at about 500x magni-
fication. A, Neohelix major (Binney), ANSP uncataloged, 505x. B, Mesodon normalis (Pilsbry), ANSP un-
cataloged, 500x. C, Xolotrema fosteri (F. C. Baker), ANSP 117483, 510x. D, Patera laevior (Pilsbry), ANSP
186465, 500x. E, Triodopsis fallax (Say), ANSP 192768, 550x. F, Inflectarius inflectus (Say), ANSP 91616,
ee G, Allogona profunda (Say), ANSP 77867, 520x. H, Appalachina sayana (Pilsbry), ANSP 264654,
5%:
72 EMBERTON
FIG. 4. The basal denticles or basal regions of the same specimens as Fig. 2, at about 500x magnification.
A, Neohelix major (Binney), ANSP uncataloged, 515x. B, Mesodon normalis (Pilsbry), ANSP uncataloged,
500x. C, Xolotrema fosteri (F. C. Baker), ANSP 117483, 520x. D, Patera laevior (Pilsbry), ANSP 186465,
510x. E, Triodopsis fallax (Say), ANSP 192768, 540x. F, Inflectarius inflectus (Say), ANSP 91616, 540x. G,
Allogona profunda (Say), ANSP 77867, 510x. H, Appalachina sayana (Pilsbry), ANSP 264654, 520x.
POLYGYRID SHELL MICROSCULPTURE 73
FIG. 5. The body-whorl sculptures of the same specimens as Fig. 2, at about 100x magnification. A,
Neohelix major (Binney), ANSP uncataloged, 102.0x. B, Mesodon normalis (Pilsbry), ANSP uncataloged,
101.5x. C, Xolotrema fosteri (F. C. Baker), ANSP 117483, 104.54ts. D, Patera laevior (Pilsbry), ANSP
186465, 102.5x. E, Triodopsis fallax (Say), ANSP 192768, 99.5x. F, Inflectarius inflectus (Say), ANSP 91616,
102.5x. G, Allogona profunda (Say), ANSP 77867, 104.0x. H, Appalachina sayana (Pilsbry), ANSP 264654,
102.5x.
74 EMBERTON
only slight Convergence between Al. pro-
funda and Ap. sayana (basal node much
broader in Al. profunda, parietal denticle
lacking in Al. profunda. Convergences be-
tween iterated pairs are consistently close in
overall shell size and shape, and apertural
size and shape (Fig. 2).
Flat Shell Forms: On the parietal denticle or
parietal region, convergences disintegrate at
high magnification (Fig. 3), with the notable
exception of X. fosteri and P. laevior, both of
which have a smooth surface dotted with low
mounds bearing wide, shallow, rough-bot-
tomed craters; the mounds are virtually iden-
tical between species in their sizes, densities,
apparently random distributions, and struc-
tural details (Fig. 3c, d). This microstructural
convergence is all the more remarkable given
the great variation in this region among the
other six species. Background surfaces
range from smooth (Fig. 3b, e, g, h) to floc-
culent (Fig. 3a) to randomly pitted (Fig. 3f).
Secondary structures range from large,
straight escarpments both sparse (Fig. 3a)
and dense (Fig. 3g), to small dense escarp-
ments both straight (Fig. 3b) and polygonal
(Fig. 3e), to rounded pustules (Fig. 3f), to
shallow canals that are randomly sized and
directed (Fig. 3h).
On the basal denticle or basal region, mi-
crosculpture in each sympatric-convergent
species (Fig. 4) is similar to that on its parietal
denticle or region, with the exceptions of X.
fosteri and P. laevior. These two species
share, on a smooth background, a pattern of
polygonal escarpments (Fig. 4c, d) that are
similar in density and dispersion to, but very
different in structure from, the cratered
mounds they share on the parietal denticles
(Fig. 3c, d). These polygonal escarpments are
similar in size and shape, but are slightly
more uptilted in P. laevior than in X. fosteri.
The other six species have virtually identical
backgrounds on their basal and parietal den-
ticles or regions, except that the flocculence
in Figure 4a is less pronounced than in Figure
3a. In secondary structures, Figures 4f and 3f
are virtually identical, as are Figures 4e and
3e, and Figures 4b and 3b. Figure 4a is sim-
ilar to Figure 3a, but with the significant ad-
dition of a new structure: a smooth-surfaced
puddle overlying a low, bumpy knob. Figure
4h has the canals of Figure 3h, but they are
much smaller and sparser, and there is the
addition in Figure 4h of low mounds. Figure
4g differs from Figure 3g only in that its es-
carpments are generally slightly shorter and
less straight.
Globose Shell Forms: The shell body-whorl
surface at high magnification (Fig. 5) shows
additional features previously undetected.
The microsculptural convergence between
Neohelix major and Mesodon normalis, indis-
tinguishable under the dissecting micro-
scope at 50x (Emberton, 1995a), is readily
detectable under SEM. Although both have a
matte-like surface produced by a pattern of
large transverse ridges crossed by smaller
spiral cords, the cords in N. major (Fig. 5a)
are even in width, whereas the cords in M.
normalis are variable in width and generally
much wider than in N. major. Both have un-
even, transverse micro-wrinkles, which are
more pronounced in N. major, however. Fur-
thermore, N. major has an even pattern of
parallel, spiral microstriae that are totally
lacking in M. normalis.
These spiral microstriae appear in all three
members of the Triodopsinae (Fig. 5a, c, e),
as well as in Allogona (Fig. 5g), but in none of
the four members of the Mesodontini (Fig.
5b, d, f, h). The microstriae are periostracal
structures only, as they do not appear in the
underlying calcium carbonate layer in those
patches where the periostracum has flaked
otf (Fig 5a, 6; 0):
Tridentate Shell Forms: Periostracum also
seems to be the sole source of the unique
pattern of jumbled wrinkles or folds that tend
toward a Spiral direction in /nflectarius inflec-
tus (Fig. 5f), the hairs of which (broken in this
specimen) are simple outward extensions of
arcuate periostracal folds, as well as of the
unique network of transverse-trending wrin-
kles in Xolotrema fosteri (Fig. 5c). The spirally
oriented pustules of Patera laevior, on the
other hand, are also part of the calcium car-
bonate shell matrix, as evidenced by the pus-
tules over which the periostracum had flaked
off (Fig. 5d). The same seems to be true of
the unusual nodulose spiral cords of Allog-
ona profunda (Fig. 59).
The remaining outstanding features of
comparative shell body-whorl microsculpture
in Figure 5 are the transverse ridges, which
vary in size, shape, density, and angle. In the
convergent pair N. major and M. normalis
(Fig. 5a, b), they are equally large (only one
complete ridge appears in each photograph),
low, and rounded in profile, but in N. major
they are slightly more angled from the vertical
POLYGYRID SHELL MICROSCULPTURE 73
than in M. normalis. In the convergent pair X.
fosteri and P. laevior (Fig. 5c, d), the trans-
verse ridges are about equal in angle, size,
and density (three ridges in each photo-
graph), but they are extremely weak in P. lae-
vior. The shell convergence between 7. fallax
and /. inflectus breaks down entirely at mi-
crosculptural level (Fig. 5e, f): Т. fallax has
strong, dense, well-separated, sharply an-
gled transverse ridges, whereas /. inflectus
nearly lacks them entirely. The transverse
ridges of Al. profunda (Fig. 5g), although
equal in density and angle to those of N. ma-
jor (Fig. 5a), are about twice as broad, so
broad in fact that they are adjacent, sepa-
rated by only narrow gutters. Thus, they are
quite different from the convergent Ap. say-
ana, whose pronounced, narrow, well sepa-
rated, mildly angled ridges are more like a
stronger version of P. laevior (Fig. 5d).
Тре N. dentifera—l. ferrissi Convergence
The extreme shell-shape convergence be-
tween Neohelix dentifera and Inflectarius fer-
rissi (Fig. 6a, b; Emberton, 1991b) carries
strong clues of its origins in its apertural and
body-whorl microsculptures (Fig. 6c-h). In
the case of N. dentifera, these clues conflict
slightly with anatomical and allozymic evi-
dence (Emberton, 1988, 1991b). Thus, al-
though N. dentifera’s parietal denticle (Fig.
6c) shares a flocculent-textured background
surface with its congener N. major (Fig. 3a),
the dominant sculpture of short, polygonal
escarpments is much closer to that of the
related Triodopsis fallax (Fig. 3e), from which
it differs only in the much steeper angles of
its escarpments. The discrepancy is similar
for N. dentifera’s basal apertural region (Fig.
6e), which has a flocculent ground similar to
that of N. major (Fig. 4a), but has a sculpture
of steep, polygonal escarpments which are
similar in density and distribution to those of
Xolotrema fosteri (Fig. 4c) and which are sim-
ilar in their small size and steep polygons to
those of Triodopsis fallax (Fig. 4e). In the dis-
tinctly exaggerated degree of their steep-
ness, however, the basal-lip polygons of
Neohelix dentifera are convergently more
similar to Patera laevior (Fig. 4d). Despite
these discrepancies, apertural microsculp-
ture clearly agrees with nonconchological
data (Emberton, 1988) in placing N. dentifera
in the Triodopsini. Body-whorl microsculp-
ture, on the other hand, is entirely concor-
dant: N. dentifera (Fig. 6g) is nearly identical
to its congener N. major (Fig. 5a), from which
it differs only in its broader spiral cords and
weaker transverse wrinkles.
The phylogenetic affinities of /. ferrissi (Fig.
6b) based on shell microsculpture are clear
and concordant with anatomical and allozy-
mic data (Emberton, 1991a, b). Its parietal
denticle has the same randomly pitted back-
ground surface and the same rounded pus-
tules as I. inflectus (Fig. 3f), except that the
pustules are arrayed in unevenly parallel rows
instead of distributed randomly as in /. inflec-
tus. Inflectarius ferrissis basal apertural re-
gion (Fig. 6f) also seems homologous with
the basal denticle of /. inflectus (Fig. 4f), with
a similar randomly pitted background surface
(less evident in Fig. 6f due to low contrast),
and with similar rounded pustules, which are
more sparsely distributed in /. ferrissi. The
unique body-whorl microsculpture of /. fer-
rissi (Fig. 6h) consists of the same jumbled
pattern of spiral-trending wrinkles as in /. in-
flectus (Fig. 5f), but on a finer and lower size
scale.
Thus, shell body-whorl microsculpture
and, to a lesser extent, shell apertural micro-
sculpture tend to bear out generic relation-
ships in Neohelix and Inflectarius, despite ex-
treme divergences in shell size and shape.
Divergences Within the Flat-Shell Clades
Intrageneric divergence in body-whorl mi-
crosculpture is extreme in Xolotrema (Figs.
5c, 7) and in Patera (Figs. 5d, 8), with struc-
tural homologies difficult if not impossible to
decipher. Nevertheless, these two genera,
which closely converge on each other in shell
size and shape (Figs. 2c and 2d; Figs. 7a and
8a) and in apertural microsculpture (Figs. 3c
and 3d; Figs. 4c and 4d), are always clearly
distinguishable in body-whorl microsculp-
ture, as discussed below.
The Xolotrema Clade: Shell divergence be-
tween the presumably hybridizing sister spe-
cies Xolotrema obstricta and X. denotata is
extreme, not only on a gross scale (Fig. 7a,
b), but also at high magnification (Fig. 7c, d)
and at very high magnification (Fig. 7e, f), at
which they also can be seen to diverge from
their congener X. fosteri (Fig. 5c). Xolotrema
obstricta’s peripheral keel and transverse rib-
bing are lacking in X. denotata, which bears
periostracal hairs (all broken in this speci-
men) lacking in X. obstricta. Xolotrema fosteri
lacks all these features, except transverse
76 EMBERTON
FIG. 6. Two conchologically convergent species that are also are ecologically parallel and convergent
(Emberton, 1991b). А, С, E, G, Neohelix dentifera (Binney), ANSP 90119: A, aperture, 4.56%, С, parietal
denticle, 540x; E, basal apertural lip, 505x; G, body-whorl sculpture, 104.0x. B, D, F, H, Inflectarius ferrissi
(Pilsbry), ANSP 98085: B, aperture, 4.56x; D, parietal denticle, 505x; F, basal apertural lip, 510x; H,
body-whorl sculpture, 102.5x.
POLYGYRID SHELL MICROSCULPTURE 77
FIG. 7. Two conchologically divergent but hybridizing sister species. A, C, E, Xolotrema obstricta (Say),
ANSP 68553: A, body whorl to the left of the aperture, 6.25x; C, body-whorl sculpture, 39.0x; E, body-whorl
sculpture, 101.0x. B, D, F, Xolotrema denotata (Férussac), ANSP 172721: B, body whorl to the left of the
aperture, 6.3x; D, body-whorl sculpture, 38.7x; F, body-whorl sculpture, 104.0x.
ribs, which are nonetheless lower, denser,
and more angled than those of X. obstricta.
Xolotrema fosteri’s spiral microstriae are en-
tirely lacking in both X. obstricta and X. de-
notata. The strongest candidate for homol-
ogy among the three species is in the
periostral wrinkles, but these vary enor-
mously. In X. fosteri, the wrinkles are small
and appear only sporadically in dense retic-
ulate patterns oriented transversely in the
gullies between transverse ribs. In X. ob-
stricta, the wrinkles are very large and appear
universally in a dense, somewhat reticulate
pattern oriented at an angle between trans-
verse and spiral. The wrinkles in X. denotata
are medium to large and universally distrib-
uted in a sparse, somewhat reticulate pattern
generally oriented transversely, and regularly
punctuated at right angles by short, thick
hairs that form cross patterns on locally
thickened transverse wrinkles. Thus, these
three types of wrinkles share a generally re-
ticulate pattern, but even in this factor, their
differences are so great (Figs. 5c, 7e, 7f) as to
78 EMBERTON
FIG. 8. A species convergent on and sympatric with Xolotrema obstricta (Fig. 7a, c, e), and two of its
congeners. A, B, D, Patera sargentiana (Johnson 4 Pilsbry), ANSP 150249: A, body whorl to the left of the
aperture, 6.30x; B, body-whorl sculpture, 39.0x; D, body-whorl sculpture, 101.0x. C, Patera perigrapta
(Say), ANSP 160543, body-whorl sculpture, 104.5x. E, Patera appressa sculptior (Chadwick), ANSP
128954, body-whorl sculpture, 102.5x.
defy detection of homology. In fact, the peri-
ostracal wrinkles of X. denotata (Fig. 7f) are
much more similar to those of /nflectarius in-
flectus (Fig. 5f), a close convergence that dif-
fers slightly in the general orientation of the
wrinkles (spiral in /. inflectus and transverse in
X. denotata) and that differs greatly in the
form of periostracal hairs (broad upward ex-
tensions of arcuate folds in /. inflectus; nar-
row, buttressed blades at right angles to
straight folds in X. denotata). The perio-
stracum is quite thick in X. denotata, as evi-
denced by the depth of the cracks appearing
in Fig. 7f).
The Patera Clade
The intersubfamilial convergence in sym-
patry between Patera sargentiana (Fig. 8a, b,
d) and Xolotrema obstricta (Fig. 7a, C, e) is
rather close. Both have large, flat, heavily
ribbed shells with peripheral angulations
POLYGYRID SHELL MICROSCULPTURE 19
(Figs. 7a, 8a), but in X. obstricta the angula-
tion is a pronounced keel. The transverse ribs
are equal in density and form (Figs. 7c, 8b),
but are more raised in P. sargentiana. Both
have spirally oriented ridges (Figs. 7e, 8d),
but in X. obstricta these are periostracal wrin-
kles or folds in a vaguely reticulate pattern,
whereas in P. sargentiana they are shell-ma-
trix pustulose cords in regularly parallel pat-
tern, with an additional underlying substruc-
ture of weak transverse cords.
Patera sargentiana's parallel spiral rows of
micro-pustules (Fig. 8d) are clearly homolo-
gous with those of P. laevior (Fig. 5d) de-
scribed above. Another congener, P. peri-
grapta (Fig. 8c), lacks pustules entirely, but
bears parallel spiral grooves equal in density
to the pustular rows of P. laevior. A fourth
congener, P. appressa sculptior (Fig. 8e),
combines the grooves of P. perigrapta (but
weaker than in that species) with the pustules
of P. laevior (but stronger than in that spe-
cies): its microsculpture is one of pustules
equally spaced within shallow, parallel, spiral
grooves. Thus, it appears that spiral grooves,
spiral rows of pustules, and spiral cords of
pustules are all homologous in Patera, and
concomitantly that the spiral gullies in P. sar-
gentiana (Fig. 8d) are not homologous with
the spiral grooves of P. perigrapta (Fig. 8c).
Transverse ribbing in P. laevior, P. perigrapta,
and P. appressa sculptior is similarly weak
and variable, entirely unlike the strong ribs of
P. sargentiana, but possibly homologous
with the secondary, weak transverse ribs of
that species.
Shell Hairs: Convergence and Divergences
Convergent periostracal hairs were de-
scribed and compared above for Inflectarius
inflectus (Fig. 5f) and Xolotrema denotata
(Fig. 7f), although incompletely because the
hair tips were broken off in both specimens.
The periostracal hairs of Vespericola colum-
biana pilosa, which are shown unbroken and
at the same high magnification in Figure 9a,
are entirely different in structure. These hairs
are thick, rigidly curved, and columnar, aris-
ing from shallow, socket-like depressions,
and are relatively unbuttressed. They arise
from a unique background surface sculpture
of both spirally and transversely oriented pat-
terns of periostracal wrinkles, punctuated by
large but weak transverse ribs and by small
shelf-like protrusions (Fig. 9a).
In Stenotrema (Figs. 9c, e; Fig. 10d), the
periostracal hairs and surface sculptures of
the two examined species are so different
that they seem entirely non-homologous.
Stenotrema maxillatum (Fig. 9c) has relatively
small, dense, regularly arranged, backward-
directed, elongate-conic hairs that are
slightly buttressed transversely and that are
marked by short, forward, spiral wrinkles in
an otherwise transversely wrinkled, smooth
background surface. Stenotrema barbigerum
(Fig. 9e), on the other hand, bears relatively
large, moderately dense, regularly arranged,
transverse folds that arc with the concave
side forward, that have tiny, low spines on
their forward surfaces, and that lie in a back-
ground surface of minute, densely packed,
parallel, tranverse ridges overlying an uneven
system of shallow, spiral grooves. The con-
spicuous, micro-spinose, transverse folds of
S. barbigerum vary in length and shape, de-
pending on position (Fig. 10d): on the upper
shell whorls they are low in profile, on the
lower shell whorls their central regions are
drawn outward and backward into thorn-like
hairs, and on the shell's keeled periphery
they extend outward into long, unevenly
blade-like hairs (Fig. 10d; Fig. 9e: upper
right).
Unlike in Stenotrema, homologies among
periostracal hairs of the genus /nflectarius
(Figs. 5f; 6h; 9b, d, f) are much more evident,
despite extensive morphological radiation.
The hairs of /. subpalliatus (Fig. 9f) seem to
be enlarged versions of the hairs described
above of I. inflectus (Fig. 5f): both are long,
arcuate, high-standing folds rising from a
background of smaller, variously oriented
wrinkles. Inflectarius subpalliatus’s sister
species, /. ferrissi (Fig. 6h), lacks hairs en-
tirely, and carries only vestigial traces of folds
and wrinkles in its relatively featureless body-
whorl microsculpture. In stark contrast to this
effacement, the hairs of /. magazinensis (Fig.
9b) show increased complexity: the bottom
of the arcuate fold is abruptly curled forward
in a scoop-like fashion, the central high-point
of the fold is thickened and curled over like a
cresting wave, and extending from this crest
is a downward arching secondary fold that
continues onto the background surface as a
rear-support buttress. These same modifica-
tions are developed even further, to a re-
markable degree, in /. smithi (Fig. 9d). In this
species, the entire arcuate fold is relatively
deeply arched and scoop-like; the central
high-point extends forward as a long, blunt,
club-like structure with a surface sculpture of
80 EMBERTON
FIG. 9. More periostracal hairs (body-whorl microsculpture). A, Vespericola columbiana pilosa (Henderson),
ANSP 158355, 100.0x. C, Stenotrema maxillatum (Say), ANSP 170141, 99.0x. E, Stenotrema barbigerum
(Redfield), ANSP 170110, 102.0x. B, Inflectarius magazinensis (Pilsbry & Ferriss), ANSP 395865, 102.5x. D,
Inflectarius smithi (Clapp), ANSP 160055, 100.5x. F, /nflectarius subpalliatus (Pilsbry), ANSP 171134,
103.0x.
minute, regular, adjacent pits; and the sec-
ondary, downward-arching, rear-buttress
fold is high-standing and strongly developed.
Character Analysis
All of the ten species that were examined
for apertural microsculpture have patterns of
escarpments, nodules, or mounds (their ab-
sence in Fig. 3h is considered an artifact of
the sparse distributions of mounds in that
species: Fig. 4h). These microprojections
seem to be homologous, with a basic mor-
phology of inclined, crystalline platelets (Figs.
3e, 4c-e, 6c, e) that is modified by various
coating surfaces. A good example is the
highly modified surface of the parietal denti-
cle of Inflectarius ferrissi (Fig. 6d), which
shows little evidence of crystalline platelets.
At lower magnification, however, this surface
can be seen to coat only the leading edge of
the parietal denticle (Fig. 10a), the uncoated
interior of which has a standard pattern of
microplatelets (Fig. 10c). Similarly, in the
closely related /. inflectus, the rounded nod-
POLYGYRID SHELL MICROSCULPTURE 81
FIG. 10. A, C, apertural parietal denticle of Inflectarius ferrissi (Pilsbry), ANSP 98085: A, tip region, 43.05x;
C, central region, 515x. B, edge of apertural parietal denticle of Inflectarius inflectus (Say), ANSP 91616,
127.5x. D, mid region of the shell of Stenotrema barbigerum (Redfield), ANSP 170110, 12.35x.
ules (Fig. 3f) are seen at a lower magnifica-
tion to be crystalline microplatelets as they
lose some of their coating away from the
edge of the parietal denticle (Fig. 10b).
Similar crystalline microprojections have
been found in a wide variety of pulmonates,
and in other gastropod groups as well
(Solem, 1970, 1972, 1973; Solem 4 Lebryk,
1976). They seem therefore to be construc-
tiona! aspects of apertural deposition (Wilbur
8 Saleuddin, 1983; Watabe, 1988), although
it has been argued that in some cases their
shape is modified by natural selection for de-
fense (Solem, 1972).
The relative sizes and distributions of mi-
croprojections seen in this study do not seem
to be reliable systematic characters, because
they correlate with shell size. Thus, three of
the four large shells (Fig. 2a, g, h) have the
largest and sparsest microprojections (Figs.
3a, 9, h; 4a, g, h); the two small shells (Fig. 2e,
f) have the smallest and densest micropro-
jections (Figs. 3e, f; 4e, f); and the four inter-
mediate-sized shells (Figs. 2c, d; 6a, b) and
one of the large shells (Fig. 2b) have micro-
projections that are intermediate in size and
density (Figs. 3b-d; 4b-d; 6c-f).
Based on this analysis, a single, multi-state
character can be proposed:
Character 1. Apertural coating.
State a. No coating on parietal or basal
denticle/region; surface very smooth and
featureless; microprojections crystalline and
clean. Triodopsis fallax.
State b. Thin, flocculent coating on both
parietal and basal denticles/regions; surface
flocculent; microprojections clean to partially
coated. Neohelix major and N. dentifera.
State b’. Same as state b, but on the basal
region, the microprojections and their sur-
roundings have an additional coating of thin,
smooth material. N. major.
State c. Smooth-surfaced coating on the
parietal denticle/region only; flanks but not
tips of parietal microprojections coated; no
coating on basal denticle/region. Xolotrema
fosteri and Patera laevior.
State d. Medium-thick, minutely pitted
coating on both parietal and basal denticles/
regions; microprojections entirely covered on
denticle edges, but with tips exposed away
from denticle edges. Inflectarius inflectus and
|. ferrissi.
82 EMBERTON
State d’. Same as state d, but the coating
covers only the edge of the parietal denticle,
where it is thick and forms parallel rows. /.
ferrissi.
State e. Thick coating scored with shallow
canals of random size and orientation, on
both parietal and basal denticles/regions; mi-
croprojections thickly and entirely covered.
Appalachina sayana.
State f. Thin, smooth coating on both pa-
rietal and basal denticles/regions; micropro-
jections completely to partially covered. Me-
sodon normalis and Allogona profunda.
Analysis of body-whorl microsculpture is
based on the 21 species studied. Intrage-
neric variation is so great in transverse ribs
that they seem unreliable as systematic char-
acters. The same must be said for the pat-
terns of (but not necessarily for the presence
of) periostracal wrinkles. The periostracum
forms as a flexible, curtain-like sheath that
later is sclerotized by quinone tanning
(Saleuddin & Petit, 1983; Waite, 1983), so the
pattern of wrinkles may be influenced by a
number of environmental and constructional
factors other than phylogenetic constraints.
Periostracal hairs are always associated (in
this sample) with periostracal wrinkles, how-
ever.
The following characters seem reliable.
Character 2. Spiral microstriae.
State a. Present. Neohelix major, N. dentif-
era, Xolotrema fosteri, Triodopsis tridentata,
Allogona profunda.
State b. Absent. All other examined spe-
cies.
Character 3. Spiral cords, smooth unless tra-
versed by spiral microstriae.
State a. Present. Neohelix major, N. dentif-
era, Mesodon normalis.
State b. Absent. All other examined spe-
cies.
Character 4. Spiral cords, nodulose.
State a. Present. Allogona profunda.
State b. Absent. All other examined spe-
cies.
Character 5. Spiral grooves/pustules/pustu-
late cords.
State a. Present. Patera laevior, P. sargen-
tiana, P. perigrapta, P. appressa.
State b. Absent. All other examined spe-
cies.
Character 6. Periostracal wrinkles.
State a. Present. Xolotrema fosteri, X. ob-
stricta, X. denotata, Vespericola columbiana,
Stenotrema maxillatum, $. barbigerum, In-
flectarius inflectus, |. ferrissi, |. smithi, I. mag-
azinensis, |. subpalliatus.
State b. Absent. All other examined spe-
cies.
Character 7. Periostracal hairs.
State a. Thin, straight, cruciform base. Xo-
lotrema denotata.
State b. Thick and round, recurved,
socket-like base. Vespericola columbiana.
State c. Thick, straight, simple base.
Stenotrema maxillatum.
State d. Thick to thin, straight to curved,
long arcuate sculpted base. Stenotrema bar-
bigerum.
State e. Thin, curved, long arcuate smooth
base. Inflectarius inflectus, |. subpalliatus, 1.
magazinensis, |. smithi.
State e”. Same as state e, but with arched
medial buttress and thickened central exten-
sion. Inflectarius magazinensis, |. smithi.
State e”. Same as state e”, but with medial
buttress very large and central extension very
long, clubbed, and sculpted. /. smithi.
State f. Absent. All other examined spe-
cies.
Figure 11 maps the informative character
states onto the phylogenetic hypothesis pre-
viously shown in Figure 1. There are 12 infor-
mative character states, of which seven ap-
pear homoplastic in Figure 11. One of these
homoplasies (state 7e’) is spurious, because
it actually resolves a trichotomy by providing
a new synapomorphy uniting /nflectarius
magazinensis with 1. smithi. Two of the ho-
moplasies microsculpturally enhance the
general shell convergences between X. fos-
teri and P. laevior (state 1c) and between N.
major and M. normalis (state 3a). The ho-
moplasies in state 6a involve multiple origins
of wrinkles in the periostracum, yet this state
is still informative in uniting the three species
of Xolotrema, for example. The loss of peri-
ostracal hairs in /. ferrissi (state 7f) accompa-
nied its great evolutionary shifts in shell size
and in ecology (Emberton, 1991b). The re-
maining homoplasies involve the apertural
coatings of Al. profunda and M. normalis
(state 1f) and the spiral microstriae of most
triodopsins and Al. profunda (state 2a). Thus,
six of the twelve informative microstructural
character states (1b, 1d, 2b, 5a, 7e, 7e’) sup-
POLYGYRID SHELL MICROSCULPTURE 83
port the phylogenetic hypothesis without ho-
moplasy.
DISCUSSION
Although this study is preliminary, it offers
hope that the shells of polygyrids—and by
inference the shells of other land-snail fami-
lies—are not entirely useless for hypothesiz-
ing phylogeny. Thus, although polygyrid
gross shell morphology and ontogeny
yielded virtually no phylogenetic resolution
among subgenera (Emberton, 1995b: fig. 16),
polygyrid microsculptural shell morphology
has so far yielded potential new informative
characters with a 50% (6 of 12) “success
rate” in resolving a previously, robustly hy-
pothesized phylogeny (this paper: Fig. 11).
Verifying these characters will require much
more work, which will also undoubtedly dis-
close many new microsculptural characters.
Of the new characters tentatively pro-
posed, some are particularly intriguing. Spiral
microstriae, a possible new synapomorphy
for the tribe Triodopsini, may finally provide a
means of distinguishing fossils of this tribe
from those of the iteratively convergent tribe
Mesodontini (Emberton, 1994a, 1995b). The
homology among adult-shell spiral pustules,
spiral pustular ridges, and spiral grooves pro-
posed here for Patera is extreme, but is in line
with Pilsbry's (1940: 576) remark that, in the
embryonic sculpture of many polygyrid spe-
cies, “many stages in the transition from
striae to granules are found.”
The remarkable radiation of periostracal
hairs in /nflectarius was unsuspected and
raises questions concerning the function of
such complex hairs as in /. smithi. Likewise,
the great discrepancy in hair microstructure
between Stenotrema maxillatum and S. bar-
bigerum raises many questions regarding the
origin(s), radiation(s), functions, and phyloge-
netic-information content of shell hairs in this
large genus, almost all species of which have
hairs.
Periostracal hair-like or scale-like pro-
cesses on the shell (Kaiser, 1966; see
Saleuddin & Petit, 1983, on the periostracum)
have evolved numerous times within the Po-
lygyridae. They evolved at least three times
within the Mesodontini alone, for example
(Emberton, 1991 a). Polygyrid shell hairs date
back to at least the Miocene (Roth & Ember-
ton, 1994) and display a wonderful variation
in size, disposition, microsculpture, and fra-
gility (Pilsbry, 1940; Solem, 1974: fig. 6; Em-
berton, 1995b: fig. 4; this paper).
Thus, polygyrids provide an excellent sys-
tem for testing functional hypotheses regard-
ing shell hairs. These hypotheses, none of
which have been tested, include the func-
tions (a) “to repel moist particles” (Solem,
1974) and prevent wet leaves from adhering
to the shell; (b) to defend against predators
(Webb, 1950); and (c) to camouflage the shell
by trapping soil and debris (Pilsbry, 1940: p.
761). Apparently, different functions are
served by different shaped hairs (Fig. 9), but
this remains to be investigated.
The different types of coatings on the ap-
erture found here were a marked addition to
previous SEM discoveries (Solem, 1970,
1972, 1973; Solem 8 Lebryk, 1976); the com-
positions and functions of these coatings are
unknown. There is evidence, however, that
coatings may change with shell age. Thus, in
Daedalochila auriformis, the parietal denticle
of a non-gerontic adult had a coating identi-
cal to that of Inflectarius inflectus (Fig. 3F),
but the parietal dentical of a gerontic D. au-
riformis was smooth and coating-free (Solem
8 Lebryk, 1976: figs. 33, 37). Although such
coatings could possibly be preservational ar-
tifacts in the form of dried mucous films, they
do not in any way resemble the mucous films
illustrated in Solem (1970: figs. 12-15).
Clearly, future studies should fully assess in-
traspecific variation in aperture microsculp-
ture if such characters are to have any value
for phylogenetics.
Two proposed microsculptural conver-
gences are remarkable for actually enhanc-
ing gross shell-form convergences in sympa-
try. Thus, Neohelix major and Mesodon
normalis (Emberton, 1994b, 1995a, 1995b:
fig. 1) also converge in their body-whorl spiral
cords, and Xolotrema fosteri and Patera lae-
vior (Emberton, 1995b: figs. 1, 17) also con-
verge in their uncoated apertures, revealing
nearly identical microsculptures of crystalline
projections from a smooth surface. Both
these pairs of species are separable, how-
ever, by other shell microsculptural features
(Fig. 11).
For future work, particularly on periostracal
hairs, it can be recommended to use very
fresh, live-collected material, preferably alco-
hol preserved. Dried periostracum can be
quite brittle, and thus no unbroken hairs
could be found on the shells of /. inflectarius
or X. denotata selected for this study. On the
positive side, however, most microstructures
84 EMBERTON
FF
—
| Tb ==
== За
| | ( = ——
|| ml 1c
|| 6al FF
== ei Ee |
| 2a | 2b ==
[| У
EA See
|
= 7 = os
| | 6a
Bl | 1f 2a
de ES
D NE |
| |
||
| | Ä г
o | 5a | | Tc
1 |
| | [PERERA
| |
| || ===
—
| | m:
| m“ 7f
| | ба] FT
|| | 7el | 7e
| | ee
| — 3 | 7е’
1d | (AA
||
|| ===
ee]
IE ==
1f
3a
Neohelix major
N. dentifera
Xolotrema fosteri
X. denotata
X. obstricta
Triodopsis fallax
Vespericola columbiana
Allogona profunda
Stenotrema maxillatum
Stenotrema barbigerum
Patera perigrapta
P. laevior
P. appressa
P. sargentiana
Inflectarius subpalliatus
I. ferrissi
I. magazinensis
/. smithi
I. inflectus
Appalachina sayana
Mesodon normalis
FIG. 11. Map of tentatively proposed microsculptural character states onto the cladogram of Fig. 1. See text
for definitions.
POLYGYRID SHELL MICROSCULPTURE 85
remained intact despite the relatively great
age of many of the specimens used in this
study. Regarding apertural microsculpture,
Solem & Lebryk (1976) found clear, uneroded
details in subfossil pupillid shells. In prepar-
ing shells for SEM, Solem (1970) cautioned
against the difficulty or impossibility of re-
moving dried mucous films, which are not a
problem in alcohol-preserved material. No
such films were noticed in this study.
Polygyrid shell-form evolution may be
unique for the sympatry of its convergences
(Emberton, 1995a, 1995b), but certainly not
for the convergences themselves, which par-
allel shell-form evolution in other stylommato-
phoran groups, such as the Helicidae sensu
lato, Bradybaenidae, and Camaenidae (Zilch,
1959-1960). For phylogenetics of these and
other land-snail groups, it can be hypothe-
sized (from this study and Emberton, 1995b)
that shell micromorphology is much more in-
formative than shell macromorphology.
ACKNOWLEDGEMENTS
Supported by Academy of Natural Sci-
ences discretionary funds and National Sci-
ence Foundation grant DEB-9201060. Caryl
Hesterman assisted on the SEM and printed
and mounted the SEM photographs.
LITERATURE CITED
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sympatric species of land snails. Venus, 47:
278-297.
ASAMI, T., 1993, Divergence of activity pattern in
coexisting species of land snails. Malacologia,
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BROOKS, D. R. & D. A. MCLENNAN, 1991, Phy-
logeny, ecology and behavior: a research pro-
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EMBERTON, K. C., 1988, The genitalic, allozymic,
and conchological evolution of the eastern North
American Triodopsinae (Gastropoda: Pulmo-
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EMBERTON, K. C., 1991a, The genitalic, allozymic,
and conchological evolution of the Mesodontini,
trib. nov. (Gastropoda: Pulmonata: Polygyridae).
Malacologia, 33: 71-178.
EMBERTON, K. C., 1991b, Ecology of a shell con-
vergence between subfamilies of polygyrid land
snails. Biological Journal of the Linnean Society,
44: 105-120.
EMBERTON, K. C., 1994a, Polygyrid land-snail
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MALACOLOGIA, 1996, 38(1-2): 87-102
ANATOMY AND SYSTEMATICS OF BUCCINANOPS GRADATUS (DESHAYES,
1844) AND BUCCINANOPS MONILIFERUS (KIENER, 1834) (NEOGASTROPODA,
MURICOIDEA) FROM THE SOUTHEASTERN COAST OF BRAZIL
Luiz Ricardo L. Simone
Museu de Zoologia da Universidade de Sao Paulo Caixa Postal 7172-01064-970
Sáo Paulo, Brazil
ABSTRACT
A morphological revision of species of the genus Buccinanops, endemic to South America,
begins with the description of B. gradatus and B. moniliferus. In an attempt to obtain data to
resolve systematic problems from the family to the specific level in this group, a detailed
anatomical description of the head-foot, pallial organs, digestive system, including odontopho-
ral muscles, and genital system are given. These animals are blind, have a vestigial valve of
Leiblein and, in the case of B. moniliferus, there is sexual dimorphism, males being about half
of the size of females.
INTRODUCTION
The systematic concepts on the South
American neogastropod species Buccinan-
ops gradatus (Deshayes, 1844) and B. monil-
iferus (Kiener, 1834) are confused at almost
every level.
There a controversy about their at the fam-
ily-level placement; some authors (e.g., Ab-
bott 8 Dance, 1983; Rios, 1994) have consid-
ered these species to be Nassariidae,
whereas others (e.g., Rios, 1985) have in-
cluded the genus in the Buccinidae. Mean-
while, Ponder (1973: 325) noted that anatom-
ical characters for the separation of these
two families have not been established.
At the generic level, B. moniliferus was
considered to belong to Dorsanum Gray,
1847, by several authors (e.g., Carcelles 8
Parodiz, 1939; Rios, 1994) and Buccinanops
Orbigny, 1841, by Calvo (1987) and Rios
(1985), based on radular characters, and by
Pastorino (1993) because of differences from
the type species of the genus Dorsanum, D.
miran (Bruguiére). Both species—B. monil-
iferus and B. gradatus—were included in the
South African genus Bullia Gray, 1834, in
early literature (e.g., Reeve, 1846) and by Ab-
bott & Dance (1983) and Allmon (1990).
At the species level, B. moniliferus in con-
trast, is well established, due its distinctive
conchological characters. Buccinanops gra-
datus, on the other hand, is a variable spe-
cies with several synonyms according to
some authors (e.g., Rios, 1975), whereas oth-
87
ers consider these synonyms to be valid spe-
cies. No convincing arguments have been
given to support either position. The available
species-group names are: В. lamarckii
(Kiener, 1834), B. cochlidius (Dillwyn, 1817),
B. uruguayensis (Pilsbry, 1897), and B. de-
formis (King & Broderip, 1832). Aggravating
these problems is the fact that neither B. gra-
datus or B. moniliferus were described with a
specific type locality.
A step in solving these systematic prob-
lems may be an anatomical analysis of well
localized and identified specimens. This pa-
per includes anatomical descriptions of Buc-
cinanops moniliferus and B. gradatus, which
will serve as the basis for future compari-
sons.
The specific names are changed to mas-
culine gender herein, following Art. 30(a)ii of
the ICZN Code for generic names ending in-
ops.
MATERIAL AND METHODS
Part of the studied material belonged to
Museu de Zoologia da Universidade de Sao
Paulo (MZUSP) and part was collected by ot-
ter trawl by fishermen in Praia Grande, Sao
Paulo, Brazil, and has been deposited in
MZUSP, fixed in 70% ethanol.
The anatomical dissections were made us-
ing standard techniques. Some anatomical
parts, such as the genital organs and anterior
region of the digestive system, were dehy-
88
drated in ethanol series, stained in carmine,
cleared and fixed in creosote. Radulae and
protoconch were also examined using SEM
in the Laboratório de Microscopia Eletrónica
do Instituto de Biociéncias da USP. All draw-
ings were made with the aid of a camera lu-
cida.
The musculature of the odontophore was
studied by means of dissection of three
specimens of each species preserved with
an extended proboscis. The jugal muscles
and peroral muscles are not described in de-
tail. For the most part, the muscles are
named according to the terminology of Wils-
mann (1942).
The synonymic list of B. gradatus is not
given here, because studies on possible syn-
onymy are continuing.
Abbreviations
anterior aorta
anterior furrow of the foot
albumen gland
siphoned anus
anterior oesophagus
auricle
mantle border
capsule gland
columellar muscle
ctenidial vein
duct to anterior digestive gland
duct of the gland of Leiblein
duct to posterior digestive gland
foot
inner gland near anus
gonopericardial duct
gill
glandular part of the kidney
gland of Leiblein
gonad
female genital pore
intestine
left cartilage
odontophoral muscles
mid oesophagus
muscular fibers
mouth
nephrostome
nephridial gland
nuchal node
nerve ring
nephridial vessel
nephridial wall
odontophore
SIMONE
oe posterior oesophagus
05 osphradium
pa posterior aorta
pc pericardic walls
pe penis
pn proboscis nerve
pp penial papilla
ps penial sinuses
pv proximal vertex of the cartilages
pw proboscis wall
ra radula
rc right cartilage
rm radular membrane
rn radular nucleus
rt rectum
sd salivary gland duct
sg salivary gland
si siphon
st stomach
SV seminal vesicle
te tentacles
ty gastric typhlosoles
uc union between both cartilages
va vas deferens aperture to pallial cavity
vd vas deferens
ve ventricle
vi valve of Leiblein
vm visceral mass
vp villous part of the kidney
Buccinanops gradatus (Deshayes, 1844)
(Figs. 1-3, 6, 9, 12-30)
Diagnosis
Shell generally homogeneous beige т
color; subsutural carina generally present,
without spines. Osphradium about 2/3 of gill
length. Radular rachidian teeth with eight
well-spaced cusps that are heterogeneous in
size; two well-developed median cusps on
lateral teeth. Odontophore with only one pair
of “m9” muscles; and with double radular
protractor muscle (m14) Both stomach
typhlosoles longitudinal. Penis long, with a
well-developed papilla. Female genital pore
papillate, surrounded by two folds.
Description
Shell: Up to 60 mm in length, homogeneous
beige, with up to 8 convex whorls (Figs. 1-3).
Protoconch of about 2.5 whorls; first whorl
glassy-smooth, semi-spherical; others with
strong axial ridges. Limit between proto-
conch and teleoconch not conspicuous. Te-
leoconch to 5 whorls; two first whorls with
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 89
FIGS. 1-11. Shells and radulae: 1, 2, dorsal and frontal view of female of Buccinanops gradatus (MZUSP
28079), scale = 10 mm; 3, frontal view of a male of В. gradatus (MZUSP 28078), scale = 10 mm; 4, 5, dorsal
and frontal view of two specimens of B. moniliferus (MZUSP 28191), scale = 10 mm; 6, radula of B.
gradatus, SEM, scale = 0.2 mm; 7, profile of the protoconch and first teleoconch whorl of B. moniliferus,
SEM, scale = 1 mm; 8, the same in apical view, scale = 0.5 mm; 9, detail of Fig. 6, scale = 0.1 mm; 10, radula
of B. moniliferus, SEM, scale = 0.2 mm; 11, dorsal view of a specimen of B. moniliferus without developed
spines on the subsutural carina (MZUSP 28181), scale = 10 mm.
axial ridges, similar to those of protoconch,
gradually disappearing on subsequent
whorls. Subsutural carina generally present,
low, rounded (Fig. 3). Periostracum very thin,
dark-brown, lost on body whorl. Aperture el-
liptic; outer lip arched, sometimes notched
by carina; inner lip concave, covered by thin
callus. Canal short, broad, bordered exter-
nally by well-developed carina.
There is considerable shell variation; the
most common form is shown in Figures 1-3,
but specimens with a shorter or taller spire
are common. The subsutural carina is lacking
in some specimens, resembling B. cochlidius
and B. uruguayensis, whereas others have a
well-developed carina and resemble B. de-
formis. The lot MZUSP 28080 has specimens
showing both conditions. Several specimens
90 SIMONE
Sl N I
FIGS. 12-14. Buccinanops gradatus anatomy: 12, visceral mass and pallial cavity organs of a female, scale
= 5 mm; 13, frontal view of a male head-foot, mantle removed, scale = 5 mm; 14, transversal section of the
mid region of the anterior oesophagus, scale = 1 mm.
have the spire without a carina and a well-
developed carina on the last whorl. No nota-
ble shell differences between males and fe-
males were found.
Operculum: Corneous, ovate-unguiculate,
with terminal nucleus, partially sealing shell
aperture. Muscle scar elliptic, near inner bor-
der. Operculum deformation very common.
Head-Foot: Homogeneous pale-beige in
color. Head somewhat projecting. Tentacles
long, lateral, without eyes (Figs. 13, 15). Foot
large, with furrow along anterior edge for an-
terior pedal glands (Fig. 13: af). Males with
large penis, behind right tentacle (Fig. 13).
Small posterior metapodial tentacle present.
Mantle Border: Simple, slightly thick. Siphon
developed, with smooth borders (Figs. 12,
13). Without pigment or with scanty dark
spots.
Mantle Cavity: About one whorl in length
(Fig. 12). Osphradium bipectinate, narrow,
long (about 2/3 of the total gill length), with
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 91
several leaflets on both sides. Gill monopec-
tinate, somewhat elliptic, with numerous tri-
angular, low leaflets. Hypobranchial gland a
thin glandular mass covering mantle between
gill and rectum.
Circulatory and Excretory Systems: Heart at
posterior-right side of pallial cavity (Fig. 12);
auricle fusiform; ventricle spherical, very-
large. Anterior and posterior aorta as normal
for caenogastropods (Fig. 12). Kidney large,
behind posterior-left side of pallial cavity
(Fig. 12). Internally, kidney with villous and
glandular parts (Fig. 28); nephridial gland
covering pericardial wall of kidney lumen (Fig.
28: ng). Nephrostome a slit surrounded by
muscle fibers, in mid region of kidney wall at
posterior end of pallial cavity (Figs. 12, 28:
ne).
Digestive System: Proboscis pleurembolic,
thick-muscular (Fig. 15), very-long (about
same length as shell when extended). Buccal
mass about half length of proboscis. Probos-
cis opening surrounded by thick muscular
sphincter. Mouth a vertical slit at distal end
of proboscis. Proboscis structure (Fig. 15):
odontophore in anterior half attached to inner
ventral wall; muscles at posterior odonto-
phore edge running posteriorly and attaching
to ventral half of inner proboscis surface up
to ventral face of rhynchodeal cavity (Fig. 15:
mf). Aorta, paralleled in both sides by a pair
of nerves, runs in mid line of ventral surface
covering these muscles; oesophagus lies
above all these structures, connected to pro-
boscis by tridimentional net of thin muscle
fibers.
Odontophore muscles (Figs. 20-26): (m1)
dorsal jugal muscles—origin: outer-proximal
dorsal wall of odontophore; insertion: inner-
dorsal peribuccal wall; (m2) transversal mus-
cle—uniting dorsally outer edge of both car-
tilages, involving dorsally other muscles of
odontophore; (m3) pair of lateral retractor
muscles of radula (retractor of pharynx)—or-
igin: in dorsal region of foot, running attached
to inner-ventral wall of proboscis; insertion:
proximal vertex of each cartilage (pv); (m4)
medial retractor muscle of radula—origin:
partly in dorsal region of foot, between m3
muscles, running attached to inner-ventral
wall of proboscis also between the m3, and
partly in ventral face of proximal vertex of
each cartilage (mid tensor); insertion: mainly
on ventral edge of radula; (m5) dorsal pro-
tractor muscle of radula—origin: joined with
medial retractor muscle (m4), bifurcating in
mid region of odontophore; insertion: dor-
sally on both sides of peroral wall; (m6) pair of
tensor lateral muscles—lying on anterior half
of the outer edge of both cartilages; (m7) pair
of small muscles—origin: on outer edge of
cartilages just proximal to m6 origin; inser-
tion: on peribuccal wall just proximal to m5
insertion; (m8) small muscle—origin: on outer
edges of both cartilages just proximal to m7
origin; near mid region of muscle both
branches unite for a short distance and after
they separate inserting on ventral region of
peribuccal wall near mid line; (m9) pair of
small muscles—origin: outer edge of carti-
lages just proximal to the m8 origin; insertion:
dorsal edge of radula; (m10) pair of large lat-
eral tensor muscles of radula—origin: dorsal
face of proximal vertex of cartilages; inser-
tion: mainly lateral-dorsal margin of radula,
uniting with medial retractor muscle (m4) for
about 2/3 of their length (Fig. 22); (m11)
horizontal muscle—uniting ventrally inner
edge of both cartilages; (m12) ventral jugal
muscles—origin: outer-proximal-dorsal wall
of odontophore; insertion: inner-ventral peri-
buccal wall, some muscular fibers more de-
veloped (Fig. 24); (m13) pair of large tensor
ventral muscles—origin: ventral face of pos-
terior vertex of each cartilage just at medial
retractor muscle (m4) origin; insertion: ventral
edge of radula; (m14) pair of small protractor
muscles of radula—origin: mixed with medial
retractor muscle (m4), distinguishable only
near horizontal muscle (m11); insertion: ven-
tral edge of radula between tensor ventral
muscle (m13) insertion.
Radula (Figs. 6, 9)—Rachidian flattened,
arched, with eight well-spaced cusps that are
smaller towards outer edges; lateral teeth ob-
lique, each with four cusps, marginal cusp
largest, middle two cusps smallest.
Anterior oesophagus lumen “X” in section
(Fig. 14), covered by net of radial and oblique
muscles uniting oesophagus with inner sur-
face of proboscis wall; salivary gland ducts
running on either side of oesophagus (Fig.
14: sd) and discharging into peroral chamber.
Valve of Leiblein vestigial, anterior to nerve
ring, poorly visible on outer surface of oe-
sophagus (Fig. 15), marked internally by sud-
denly change of inner longitudinal folds,
forming a low valve (Fig. 16: vl).
Two salivary glands clustered around
nerve ring (Fig. 15: sg), their ducts on outer
side of nerve ring, running to posterior half of
anterior oesophagus and within muscular net
92 SIMONE
FIGS. 15-19. Buccinanops gradatus anatomy: 15, anterior region of the digestive system and proboscis
opened longitudinally along dorsal mid line and head mid line, scale = 10 mm; 16, detail of the region of mid
oesophagus opened longitudinally, scale = 2 mm; 17, stomach in ventral view, scale = 2 mm; 18, the same
opened longitudinally, scale = 2 mm; 19, detail of the anal region, terminal region of the rectum partially
opened longitudinally to expose an inner gland, scale = 1 mm.
of anterior half of anterior oesophagus (Fig.
14). No accessory salivary glands present.
Entire oesophagus a long, somewhat uni-
form, thick muscular walled tube without
crop (Figs. 15-17); internally with several lon-
gitudinal folds (Fig. 16). Mid-oesophagus
very short (Fig. 16: me). Gland of Leiblein
long, thin, with short duct, running posteriorly
close to posterior oesophagus (Figs. 15, 16),
yellowish-brown in color.
Stomach well developed; walls somewhat
thick; two ducts to digestive glands, one dor-
sal near insertion of oesophagus, the other
ventral near opening to intestine (Fig. 17). In-
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 93
FIGS. 20-23. Odontophore of Buccinanops gradatus: 20-23, successive dissection in dorsal view. 20, only
proboscis wall opened and oesophagus removed. 21, the outer layer of muscles removed. 22, second layer
of muscles removed exposing the inner muscles. 23, most muscles removed to show the cartilages.
94 SIMONE
Ss
COCOA y
$$
ЧАСТО
(de =
a
CA E
+ nn es
ARA ai a AV:
=
FIGS. 24-26. Successive dissection in ventral view, proboscis entirely removed. 24, outer view of the
odontophore; 25, same with first layer of muscles removed. 26, second layer of muscles removed, hori-
zontal muscle (m11) opened longitudinally exposing a part of the dorsal muscles. Scales = 2 mm.
ner stomach surface rich in folds; opposite to
digestive gland ducts these folds converging;
two ventral typhlosoles present between
these ducts (Fig. 18).
Intestine thin-walled, lying anteriorly to kid-
ney (Fig. 28), in right side of pallial cavity in
males or close left side of pallial oviduct in
females (Fig. 12). Anus siphoned, slight back
of mantle border (Figs. 12, 19: an). Internally,
a sub-terminal glandular mass present (Fig.
19).
Genital System: Male. Testis in visceral mass
near columella; vas deferens initially a narrow
duct. Seminal vesicles greatly convoluted
just posterior to pallial cavity (Fig. 29). Gono-
pericaldial duct present, small (Fig. 29: gd). In
floor of pallial cavity, vas deferens a closed
duct thickened by prostate gland, except in
its posterior extremity, where there is a small
aperture (Fig. 29: va). Penis narrow, long, in-
ternally with a convoluted vas deferens and
well-developed sinuses on both sides (Fig.
29); tip rounded, with a small pointed papilla
on right side in which the vas deferens opens
(Figs. 13, 29).
Female. Ovary in visceral mass mixed with
digestive gland, mainly concentrated near
columella. Oviduct extremely narrow, on right
side of pallial cavity, with well-developed al-
bumen-capsule glands (Fig. 27), both difficult
to differentiate from one another, occupying
about half of pallial cavity length (Fig. 27).
Vestibule thin-walled, somewhat long. Fe-
male genital aperture papillated, surrounded
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 95
FIGS. 27-29. Buccinanops gradatus anatomy: 27, detail of the right side of the pallial cavity in inner view
to show the pallial oviduct; 28, kidney chamber opened ventrally; 29, detail of a cleared penis and right side
of the pallial cavity floor showing the mid and anterior regions of the male reproductive system. Scales =
2 mm.
by two folds, right fold thin, left fold larger
broad (Fig. 30), sited in the posterior-right
side of anus (Figs. 12, 27, 30).
Habitat. Sandy-mud bottoms, from 5 to 25
m depth. For data on posture and capsules,
see Penchaszadeh (1973).
Range. With certainty from Rio de Janeiro
to Sao Paulo coast; specimens from other
regions still under study.
Examined specimens. BRAZIL, otter trawl.
Rio de Janeiro: MZUSP 28184, 1 specimen,
Cabo de Sao Thomé (11/ii/1969); MZUSP
15295, 1 specimen, Atafona, Sao Joao da
Barra. Sao Paulo: Ubatuba: MZUSP 28080, 8
specimens, Itaqua Beach (i/1971, Mon-
touchet col.); MZUSP 28185, 2 specimens,
Cabras Is., Anchieta Is. (28/vi/1978); MZUSP
28186, 2 specimens, Anchieta Is. (4/vili/1960,
96 SIMONE
FIG. 30. Buccinanops gradatus anatomy: detail of
Fig. 27, showing the female genital pore, scale = 1
mm.
Clarimundo col.); MZUSP 28081, 1 speci-
men, same (21/4/1979). Baixada Santista:
MZUSP 28183, 5 specimens, from Barra de
Santos to Guarujá (vii/1969, Instituto de
Pesca col.); MZUSP 28192, 20 specimens,
Perequ& Beach, Guarujá (6/vi/1985); MZUSP
28193, 8 specimens, Santos Bay (2/1x/1970);
MZUSP 28187, 2 specimens, from Moela Is.
to Ponta Perequê (17/v/1962, Clarimundo
col.); MZUSP 28188, 5 specimens, Goes
Beach, Guarujá (17/vili/1970, Colella col.);
MZUSP 28189, 2 specimens, from Barra de
Santos to Farol da Moela (vii/1969, Instituto
de Pesca col); MZUSP 28078, 28079,
28082, 25 specimens, Barra de Santos (21/
ix/1970, Colella col.); MZUSP 28183, 14
specimens, Моей Is., 15 т deep (17/v/1962,
Clarimundo col.). Praia Grande, off Bo-
queirao Beach: MZUSP 28190, 12 speci-
mens (i/1994, Simone col.); MZUSP 27319,
12 specimens (10/1/1990, Simone col.). Total:
117 specimens.
Buccinanops moniliferus (Kiener, 1834)
(Figs. 4, 5, 7, 8, 10, 11, 31=42)
Buccinum moniliferum Kiener, 1834: 2, pl. 3,
fig. 8; Reeve, 1842: 234, pl. 268, fig. 4;
Deshayes, in Lamarck, 1844: 191.
Bullia armata Gray, 1854: 26; Reeve, 1846: pl.
1, fig. 2 [Hab. ?]; Adams 4 Adams, 1858:
113; Kobelt, 1877: 290; Tryon, 1882: 14,
pl. 6, figs. 82, 83; Paetel, 1888: 116;
Morretes, 1949: 98.
Buccinanops moniliferum: Orbigny, 1845:
199; Rios; 1985: 103, pl 35/1ig 456:
Calvo, 1987: 143, fig. 122 (radula); Pas-
torino, 1993: 160-165, figs. 1-3 (radula).
Buccinum (Buccinanops) maniliferum: (err.)
Orbigny, 1846: 434.
Buccinanops cochlidium: Gray, 1854: 40
(non Dillwyn, 1817).
Buccinum armatum: Kúster, 1858: 90, pl. 15,
fig. 20.
Bullia (Buccinanops) moniliferum: Chenu,
1859: 160, fig. 750; Abbott & Dance,
1983: 117 (fig.).
Dorsanum armatum: Cossmann, 1901: 218.
Dorsanum moniliferum: Carcelles & Parodiz,
1939: 747, figs. 1, 2; Carcelles, 1944:
249; Rios, 1970: 92, pl. 28; Rios, 1975:
95, pl. 27, fig. 398; Penchaszadeh,
1971a (posture and capsules); Pen-
chaszadeh, 1971b: 480; Figueiras & Si-
cardi, 1972: 179, pl. 13, fig. 176; Mar-
{огей, 1991 (parasite); Castellanos,
1994: 89, 96, fig. 31-4 (capsule); Rios,
1994: 130, pl. 41, fig. 557.
Diagnosis
Shell generally with two spiral purple
bands on each whorl; subsutural carina with
regular-spaced spines. Osphradium about
half of gill length. Radular rachidian teeth with
nine cusps of homogeneous size; generally
only one mid cusp on lateral teeth. Odonto-
phore with two or three pairs of m9 muscles;
and with single radular protractor muscle
(m14). Typhlosoles of stomach perpendicular
one another. Male about half of female size.
Penis somewhat long, with a small node in
tip. Female genital pore single, bordered by
bulged thick muscular walls.
Description
Shell: Up to 50 mm in length, with up to
seven convex whorls, generally pale-cream,
with two broad spiral bands brown-purple on
each whorl (Figs 4, 5). Protoconch of about
2.5 whorls; first whorl smooth, others with
strong axial ridges and subsutural furrow
(Figs. 7, 8). Limit between protoconch and
teleoconch not conspicuous. First two whorls
of teleoconch with axial ridges, similar to
those of protoconch, disappearing on subse-
quent whorls. Subsutural carina present, with
short, uniform, somewhat spaced, triangular
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 97
spines turned distally and dorsally (Figs. 4, 5).
Periostracum very thin, black, lost on body
whorl. Aperture elliptic; outer lip arched,
notched by carina, with a low anal sinus; in-
ner lip concave, covered by a thin white cal-
lus. Canal short, broad, bordered externally
by well-developed carina.
Shell variation is low compared with the
preceding species, as shown in Figure 42. In
rare specimens, absence of spines in subsu-
tural carina were observed (e.g., MZUSP
28181; Fig. 11). In other specimens, there is a
homogeneous purple color, in contrast to the
common two spiral bands per whorl. Albino
and sinistral specimens are also known.
Operculum: Corneus, ovate-unguiculate,
with terminal nucleus, partially sealing shell
aperture; muscle scar elliptic near inner bor-
der. Operculum deformation very common,
rarely lost. One female (MZUSP 28151) has
two well-developed opercula side by side, in
the normal position.
Head-Foot: Homogeneous pale-beige in
color. Head somewhat projecting; tentacles
long, lateral, without eyes. Foot large, with
furrow along anterior edge for anterior pedal
glands (Figs. 31, 32: af). Small posterior
metapodial tentacle present.
Mantle Border: Simple, slightly thick (Fig.
33). Siphon developed, with smooth borders
(Figs. 31, 33), pigmented by dark-brown ir-
regular spots. Siphon with well-developed
muscular root.
Mantle Cavity: About 1.5 whorls in length
(Fig. 33). Osphradium bipectinate, narrow,
long, with several short leaflets in both sides,
lying along about half of gill length. Gill
monopectinate, elliptic, long, with numerous
triangular, low leaflets. Hypobranchial gland
thin, poorly developed, near and anterior to
anal region.
Circulatory and Excretory Systems: As de-
scribed for preceding species (Fig. 33).
Digestive System: Radular rachidian teeth
with nine cusps that are somewhat uniform in
size and close one-another (Fig. 10); marginal
teeth with only one mid cusp (Fig. 10) or
rarely with two smaller cusps, the inner cusp
longer.
In odontophore, most part of muscles and
other structures very similar to that of B. gra-
datus, except that in B. moniliferus the small
muscles originating on the outer edge of car-
tilages and inserting on the dorsal edge of
radula (called ‘‘m9”’ in preceding species) are
multiple and vary from 2 to 3 successive sim-
ilar-sized pairs. The small muscle that origi-
nates with medial retractor muscle of radula
(m14) and inserts on ventral edge of radula
near mid line is single (Fig. 34: m14a) and has
a part of its fibers inserting ventrally in
beribuccal wall also near mid line (Fig. 34:
m1 4b).
Stomach (Fig. 37) similar to that of preced-
ing species, except one typhlosoles is longi-
tudinal, from the oesophagus to the intestine
(fig. 38: ty1), whereas the other is transversal,
lying duct to posterior digestive gland (Fig.
38: ty2).
All other studied characters of the diges-
tive system of B. moniliferus are closely sim-
ilar to preceding species (Figs. 35, 36), in-
cluding characters of valve and gland of
Leiblein (Fig. 35) and anus (Fig. 33).
Genital System: Male. Testis in visceral mass
near columella. Seminal vesicles greatly con-
voluted (Fig. 39) just posterior to pallial cav-
ity. A small aperture where vas deferens en-
ters floor of pallial cavity (Fig. 39: va);
remainder closed, thickened by prostate
gland (Fig. 40). Penis narrow, long (Fig. 32),
internally a convoluted vas deferens and two
well-developed sinuses in both sides (Fig. 40:
ps); rounded tip with a very small vesicle on
right side in which vas deferens opens (Fig.
40).
Female. Ovary in columellar side of visceral
mass not mixed with digestive gland (Fig. 33).
Oviduct very narrow. Albumen and capsule
glands well developed, difficult to distinguish
from one another, occupying about half of
pallial cavity length (Fig. 41); vestibule thin-
walled, very short. Female genital aperture
small, bordered by bulged thick muscular
walls (Fig. 41). Two specimens (39.8 mm and
33.0 mm length, MZUSP 28176) have a small
node where penis occurs in males (Fig. 31).
Sexual dimorphism. Mature males notably
smaller than mature females. Mature male
length: 20.3-27.8-36.8 mm. Mature female
length: 31.0-43.5-49.5.
Habitat. Sandy-mud bottoms, from 5 to 25
m depth.
Range. From Rio de Janeiro, Brazil, to San
Matias Gulf, Argentina.
Examined specimens. BRAZIL, otter trawl.
Rio de Janeiro: MZUSP 19591, 1 specimen,
sta. IV, 22°06’S, 41°04’W, off Cabo de Sao
98 SIMONE
FIGS. 31-33. Buccinanops moniliferus anatomy: 31, frontal view of a female (MZUSP 28176) with nuchal
node, mantle partially opened, scale = 2 mm; 32, frontal view of the head-foot of a male, mantle and siphon
removed, scale = 2 mm; 33, visceral mass and pallial cavity organs of a female in inner view, scale = 10 mm.
Thome, 16 т (11/11/1969, “W. Besnard” col.).
Sao Paulo: off Ubatuba: MZUSP 28124, 17
specimens, 22°05’50”$, 41°04’12’W, 10 т
(vii/1991); MZUSP 28125, 19 specimens,
22°06’07”$, 41°04’08’W, 13 m (3/1992).
Baixada Santista: MZUSP 28179, 1 speci-
men, from Barra de Santos to Guarujá (vii/
1969, Instituto de Pesca col.); MZUSP
28181, 2 specimens, Goes Beach, Guarujá
(17/vii/1970, Colella col.); MZUSP 28084, 10
specimens, Perequé Beach, Guarujá (6/vi/
1985). Praia Grande, off Boqueiráo Beach:
MZUSP 28191, 11 specimens (i/1994, Si-
mone col.); MZUSP 26865, 2 specimens (10/
xi/1970, Ribas col.); MZUSP 28175, 20 spec-
imens (i/1994, Simone col.); MZUSP 28176,
46 specimens (i/1990, Simone col); MZUSP
27320, 2 specimens (10/1/1990, Simone col.);
MZUSP 28177, 5 specimens (Summer, 1994,
Simone col); MZUSP 28151, 86 specimens
(xii, 1991, Simone col); MZUSP 28152, 17
specimens (Summer, 1987, Simone col);
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 99
FIGS. 34-38. Buccinanops moniliferus anatomy: 34, odontophoral muscles exposed by dissection (com-
pare with the fig. 25), scale = 2 mm; 35, left view of the anterior region of the digestive system, scale = 2
mm; 36, region of the proboscis opened longitudinally in dorsal mid line, scale = 8 mm; 37, stomach in
ventral view, scale = 2 mm; 38, the same opened longitudinally, scale = 2 mm.
MZUSP 28153, 18 specimens (i/1987, Si- MZUSP 28178, 13 specimens, Prainha
mone col). Itanhaém: MZUSP 28180, 1 spec- Beach (18/1/1970, Vaz col.). Total: 271 spec-
imen, Prainha Beach (18/1/1970, Vaz col.); imens.
100 SIMONE
FIGS. 39-41. Buccinanops moniliferus anatomy: 39, mid region of the male genital duct in ventral view; 40,
dorsal view of a cleared penis and right side of the pallial floor with a detail of a section in mid region of the
penis; 41, detail of the right side of the pallial cavity showing the pallial oviduct, scales: 1 mm.
DISCUSSION
Buccinanops gradatus differs anatomically
from B. moniliferus in having: (1) the osphra-
dium proportionally longer; (2) the rachidian
teeth of the radula with fewer, more widely
spaced cusp that are less uniform in size; (3)
the lateral teeth with two well-developed in-
termediate cusps (B. moniliferus generally
has only one or two smaller cusps, see fig. 1
of Pastorino, 1993); (4) only one pair of
odontophoral ‘‘m9’’ muscles; (5) double
“m14” muscle; (6) stomach with the typhlo-
soles parallel one another; (7) absence of sex-
ual dimorphism—in B. moniliferus, the mature
male is smaller than the mature female; (8)
penis proportionally longer, and with the pa-
pilla more developed; (9) the female genital
pore in form of a small papilla surrounded by
two folds, whereas in В. moniliferus, it
bulges, has thick walls, and is without papilla.
Analysis of the anatomical characters of
other species of Buccinanops is necessary
for any systematic interpretation of the
above-cited differences. Probably, based on
number and degree of differences, both
studied species may belong to close, but dif-
ferent genera. Buccinanops moniliferus is
maintained in the genus Buccinanops, but
the generic attribution may change in future.
Pastorino (1993) gave a strong argument in
favor to the separation of this species from
ANATOMY OF BUCCINANOPS GRADATUS AND B. MONILIFERUS 101
20
10 20
FIG. 42. Graph length
circles) and 48 males (squares).
the genus Dorsanum, based on differences
from its type species, D. miran from Africa
(Allmon, 1990).
The radula of B. moniliferus is similar to
that of B. cochlidius (see Pastorino, 1993:
162, figs. 4-6), but differs in having more
cusps on the rachidian, and its largest cusp
on the right, not the left side.
Both studied species have some morpho-
logical similarity to the European Buccinum
undatum (Buccinidae) and Nassarius reticu-
lata (Nassariidae) (Fretter & Graham, 1962:
214-5, figs. 115-116), differing mainly in hav-
30 40 50
x width based оп 203 specimens of Buccinanops moniliferus, 156 females (dark
ing tentacles without eyes and by reduction of
the valve of Leiblein. The odontophoral mus-
cles of both studied species are similar to
those of Buccinum undatum (see Wilsmann,
1942), differring mainly in having: (1) the hor-
izontal muscle (m11) shorter, (2) the dorsal
protractor of the radula (m5) thinner, (3) the
lateral tensor muscle (m10) stronger, and (4)
the minor dorsal muscles (m7, 8 and 9) dif-
ferently arranged. No studies with this level of
detail of the Nassarius odontophore exists.
The ongoing comparative study on the ar-
rangement of the odontophoral muscles of
102 SIMONE
other Buccinanops and Nassarius species
may add data useful in family-level distinc-
tions.
ACKNOWLEDGMENTS
| am very grateful by Dr. Guido Pastorino,
Faculdad de Ciencias Naturales y Museo,
Universidad Nacional de La Plata, Argentina,
for detailed reading. My special thank also for
the anonymous referees for detailed revision
and criticisms.
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age dans l’Amerique Meridionale.” London. i-iil
+ 89 pp.
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sudoccidental. Apontes al conocimiento de su
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moluscos do Brasil. Arquivos do Museu Para-
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genero Buccinanops d'Orbigny, 1841 (Gas-
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Revised Ms. accepted 16 January 1996
MALACOLOGIA, 1996, 38(1-2): 103-142
SYSTEMATICS, BIOGEOGRAPHY AND EXTINCTION OF CHIONINE BIVALVES
(BIVALVIA: VENERIDAE) IN TROPICAL AMERICA: EARLY OLIGOCENE-RECENT
Peter D. Roopnarine
Department of Biology, Southeast Missouri State University, Cape Girardeau,
Missouri 63701 U.S.A.
ABSTRACT
The genus Chione ranges from the Early Oligocene of the tropical western Atlantic to the
Recent of the tropical western Atlantic and eastern Pacific. The genus is generally considered
to comprise the subgenera Chione s.s., Chionista, Chionopsis, lliochione, Lirophora, Pan-
chione, and Puberella. A phylogenetic analysis of the subgenera suggests that the genus is
paraphyletic, because its current definition excludes the related genera Anomalocardia, Pro-
tothaca and Timoclea. This problem is resolved by converting the taxonomic classification to a
phylogenetic one, constituting Puberella, Chionista, Chionopsis, lliochione and Lirophora,
newly elevated to full generic status.
The first genera to appear in the fossil record are Lirophora and Puberella in the Early
Oligocene of the western Atlantic. The genus Chionopsis appears next, in the Late Oligocene
of the western Atlantic. Chione and Panchione first occur in the Early Miocene, also of the
western Atlantic. By the Early Pliocene, Chione, Chionopsis, Lirophora and Panchione were
present in the eastern Pacific. During the Pliocene, diversities and distributions of these genera
changed dramatically. In the Early-middle Pliocene (5.2-2.5 Ma), both Lirophora and Panchione
suffered severe extinction in the western Atlantic. All supraspecific taxa present in the western
Atlantic suffered elevated extinction during the Late Pliocene. Conversely, diversities increased
in the eastern Pacific during the Pliocene, added to by the evolution of Chionista, and lliochione.
The net result is much higher Recent diversity in the eastern Pacific compared to the western
Atlantic.
Key words: Chione, extinction, Neogene, systematics.
INTRODUCTION
The reasons for diversification and extinc-
tion of a lineage are the results of complex
interactions between phenotype and envi-
ronment. While phylogenetic and morpholog-
ical analyses can lead to an understanding of
patterns of phenotypic change, the impetus
for such change must come from the organ-
ism’s biotic and abiotic environments. The
potential therefore exists for understanding
the evolution of a clade, given knowledge of
the relationships among its inclusive taxa,
and an understanding of the ecological con-
ditions under which the clade evolved.
This approach is utilized in a phylogenetic
and paleoecological study of the Cenozoic
tropical American marine bivalve genus
Chione (von Muhlfeld, 1811). Species as-
signed to this genus are among the most
abundant members of many shallow soft-
bottom communities throughout Atlantic and
Pacific tropical America (North, Central and
South), and have been so since at least the
Early Miocene. The genus has remained re-
103
stricted to tropical America since its first ap-
pearance in the Early Oligocene, and occurs
today in the Atlantic from South Carolina to
Brazil, and in the Pacific from southern Cali-
fornia to northern Peru. The purpose of this
study is to present an analysis of phylogeny,
and a taxonomic revision, of Chione subgen-
era using shell morphological characters. A
biogeographic history, focusing on the late
Neogene, is also presented using fossil oc-
currences. The geographic range of the ge-
nus is within a region that was subject to sev-
eral significant geological and oceanographic
changes during the late Neogene, notably
the uplift of the Isthmus of Panama, and the
initiation of Northern Hemisphere cooling.
These events are likely to have affected the
diversities and distributions of Chione taxa.
Many other molluscan taxa were affected
adversely (Stanley, 1986; Vermeij & Petuch,
1986; Vermeij, 1993), namely by extinction in
the Western Atlantic and subsequent restric-
tion to the tropical Eastern Pacific.
The phylogenetic analysis was performed
at the subgeneric level, because while mono-
104 ROOPNARINE
phyly of Chione is questionable (as dis-
cussed below), species within each Chione
subgenus share well-defined, discrete char-
acter states with little interspecific variation
(see, for example, Roopnarine, 1995). The
ease with which species can be assigned to
subgenera using the combination of the
states present within them argues strongly
for the monophyly of the currently defined
subgenera. The phylogenetic analysis facili-
tated a taxonomic revision of Chione, be-
cause the current taxonomy is not consistent
with the analysis. The new taxonomy will be
more accurate phylogenetically, and there-
fore offer a more accurate depiction of rela-
tionships among the subgenera.
Analyzing the distribution and diversity of
Chione subgenera revealed patterns of
change in geographic ranges. Placing the
geological and geographic ranges of the sub-
genera in a phylogenetic framework allowed
changes in biogeography and diversity to be
applied to understanding patterns of diversi-
fication and extinction. Diversity and extinc-
tion of Chione taxa were documented by ex-
amining the available records of all Chione
species of the late Neogene in both the West-
ern Atlantic and the Eastern Pacific. These
data will be used to test various hypotheses
constructed to explain the late Neogene mol-
luscan extinctions in the tropical Western At-
lantic. Prior to hypothesis testing, though, it
will be essential to present some information
explaining the situation of tropical America
during the Neogene.
Late Neogene Extinctions in
Tropical America
Tropical America during the late Neogene
was the site of dramatic oceanographic
changes, coupled with changes in faunal
composition and diversity. The two most
commonly cited causes of diversity changes
are decreasing temperature (Stanley, 1986)
and decline or disruption of planktonic pro-
ductivity levels (Vermeij, 1978, 1987; Allmon
et al., 1993; see also Jones & Hasson, 1985).
Both mechanisms, however, are plausibly
linked to two geological events, the uplift of
the Isthmus of Panama, and the initiation of
intense Northern Hemisphere cooling.
Uplift of the Isthmus of Panama may have
begun as early as the early Middle Miocene
(>15.2 mya) (Duque-Caro, 1990). Termination
of surficial circulation and final closure prob-
ably did not occur, however, until the Early
Pliocene (approximately 3.5 mya) (Coates et
al., 1992). Separation of the oceans has long
been associated with Plio-Pleistocene
changes in faunal composition in both the
tropical Western Atlantic and Eastern Pacific
(Vermeij & Petuch, 1986; Jackson et al.,
1993).
Severe late Neogene Northern Hemisphere
cooling and glaciation, documented in the
deep-sea stable isotopic record (Shackleton
8 Hall, 1985; Krantz, 1991), the stratigraphic
record of North Atlantic coastal deposits
(Krantz, 1991; Cronin, 1993) and microfaunas
(Cronin, 1991), began about 2.5-2.4 Ma
(Shackleton 8 Hall, 1985; Stanley, 1986; Cro-
nin, 1991, 1993; Allmon et al., 1993). This
event, along with later Late Pliocene-Early
Pleistocene cooling events, has been linked
to molluscan extinctions in the southeastern
United States and the Caribbean region
(Stanley, 1986). Weyl (1968) hypothesized
that closure of the Panama seaway would
have intensified the northward flowing Gulf
Stream current. The resultant change in the
heat distribution and precipitation in the
Northern Hemisphere may have initiated
the buildup of Arctic ice and the subsequent
glaciations. Therefore, the two events may be
linked.
Initial observations of the changes in faunal
composition were interpreted as а large-
scale decline in the diversity of the Western
Atlantic molluscan fauna, compared to the
Eastern Pacific (Woodring, 1966; Vermeij,
1978; Vermeij & Petuch, 1986). Recent com-
pilations of Pliocene to Recent molluscan
faunas in the Caribbean and the southeast
United States have modified this interpreta-
tion by noting that the Recent molluscan fau-
nas of the tropical Western Atlantic are actu-
ally as diverse, or more than their Pliocene
counterparts (Allmon et al., 1993; Jackson,
1993). Therefore, extinction must have been
matched by speciation and/or invasions (Ver-
meij, 1993; Vermeij & Rosenberg, 1994).
Such observations do not, however, negate
the fact that numerous molluscan taxa that
were once widespread in the tropical West-
ern Atlantic are now restricted to the Eastern
Pacific (Vermeij & Petuch, 1986), or to a few
“refuges” in the Caribbean Sea (Petuch,
1982).
A necessary step in describing the late
Neogene biological history of this region is to
explore plausible reasons why some mollus-
can taxa suffered declines in diversity in the
Western Atlantic during the Late Pliocene,
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 105
while others were either unaffected or in-
creased in diversity in the Western Atlantic or
Eastern Pacific. One possible approach to
this problem is to document the changing di-
versities within a phylogenetic context or
framework, and observe if the nature of di-
versity change is uniform throughout the
clade. Analysis of geographic and character
distributions with respect to diversity change
may subsequently yield some clues to
whether extinction was phylogenetically ran-
dom, or if it followed a character-based pat-
tern.
A basis for selective extinction is not nec-
essarily recognizable in the results of a phy-
logenetic analysis if the pattern of extinction
is related to a non-phylogenetic property
(strictly speaking, for example, geographic
distribution). This reason deserves attention
because it implies that the basis for extinc-
tion could be an environmental perturbation
with effects that transcend patterns of evo-
lutionary relationship. The two causes of ex-
tinction suggested above, cooling and de-
cline of planktonic productivity, could belong
potentially to this class of environmental per-
turbation. To demonstrate the action of this
type of variable, one could document rele-
vant physical (geological) evidence, or more
important, predict and test the effect(s) of
these agents on the organisms under study.
Tests of cooling and declining planktonic
productivity as agents of selection are ex-
plored more fully in the following sections.
Cooling and Extinction
Stanley (1984) argues that the decimation
of tropical bivalves relative to cooler-water
species would be an effect of a cooling-
based extinction mechanism. This hypothe-
sis requires that an established latitudinal
temperature gradient exists at the time of
cooling. The gradient would, prior to cooling,
have determined the development of recog-
nizable temperate and tropical faunal prov-
inces. Temperate biotas can migrate equa-
torward during times of global cooling, but
tropical biotas have no refuges. Moreover,
the provinces would presumably be compos-
ites of stenothermal and eurythermal taxa,
the differential survivals of which indicate the
occurrence of a cooling-related extinction.
For example, Stanley (1984, 1986) suggests
that the relatively higher survival of euryther-
mal bivalves from the Upper Pliocene Pine-
crest (Upper) Beds of Florida is the result
FIG. 1. Geographic ranges of Recent species of
Chione.
of a middle-Late Pliocene cooling event. The
recognition of the provinces is crucial to the
hypothesis, for only then can comparisons
be made between provinces located in dif-
ferent thermal regimes. Such a comparison
could possibly be made in the late Neogene
between the tropical Pacific and Atlantic Ga-
tunian province, and the more northerly, sub-
tropical Caloosahatchian province (Petuch,
1982; Jones & Hasson, 1985) (Fig. 1). Rec-
ognizing the provinces has depended tradi-
tionally on the identification of resident and
endemic species and their ranges (Woodring,
1966; Petuch, 1982), and inferences about
the thermal tolerances of these species
(Stanley, 1986).
The middle Pliocene (~4.0-2.5 Ma),
though not a formal stratigraphic subdivision,
is recognizable by indications of a period of
global warming following Late Miocene and
Early Pliocene cooling events (Summarized
in Cronin, 1991, and Krantz, 1991). The end
of the interval may be marked by a major
regression associated with Northern Hemi-
sphere glaciation, and quite evident in
coastal deposits of the North Atlantic, for ex-
ample in the southeast United States (Krantz,
1991). Despite data indicating that the middle
106 ROOPNARINE
Pliocene was a time of relative global
warmth, the Gatunian and Caloosahatchian
provinces were unexpectedly cooler than
other contemporaneous regions, and were
only as warm or slightly cooler than they are
today (Jones 8 Hasson, 1985; Cronin, 1991).
Gatunian waters were, however, absolutely
warmer than Caloosahatchian during this
time, by as much as 10°C in the winter, and
6°C in the summer (Cronin, 1991).
Cronin's (1991) observations do not sup-
port Stanley’s (1986) cooling hypothesis, be-
cause they imply a reduced latitudinal ther-
mal gradient in the Western Atlantic (Cronin,
1991) at a time when the hypothesis would
require a highly developed gradient. For ex-
ample, during the middle Pliocene, summer
temperatures south of Cape Hatteras, North
Carolina (Fig. 1) were approximately 2.6°C
warmer than temperatures north of the cape.
The temperature difference today is 8.6°C
(Cronin, 1991). Similarly, summer tempera-
tures differed between the southern Carib-
bean and Florida during the middle Pliocene
by approximately 3.8°C, but as much as
4.3°C today (Cronin, 1991).
The cooler temperatures in the Caloosa-
hatchian and Atlantic Gatunian regions dur-
ing atime of global warmth seem anomalous,
but it has been suggested that they could be
explained by the existence of upwelling
zones (Vermeij, 1978; Jones & Hasson, 1985;
Vermeij & Petuch, 1986; Cronin, 1991). Ac-
cording to Cronin’s (1991) data, winter tem-
peratures in the Caribbean were an average
1.1°C cooler than today, and in the Caloosa-
hatchian an average of 1.2°C cooler. The
cooling hypothesis, however, does not re-
quire that middle Pliocene temperatures be
absolutely warmer than Pleistocene and Re-
cent temperatures, simply that temperatures
declined during the time of extinction.
A simple, faunally-based test of the ex-
planatory power of the cooling hypothesis
would be to document the geographic distri-
butions of Chione subclades in the Gatunian
and Caloosahatchian provinces during the
Pliocene and post-Pliocene. The test de-
pends on the ability to distinguish between
the provinces on the basis of faunal compo-
sition (in this case excluding Chione taxa).
Petuch (1982) demonstrated that the two
provinces maintained high levels of ende-
mism, with respect to gastropods, through-
out the Early Miocene to Pleistocene. Cata-
strophic cooling would, in accordance with
Stanley’s (1984) argument, affect taxa that
were restricted to the more southerly Gatu-
nian province more severely than taxa resi-
dent in the Caloosahatchian province. In ad-
dition, one should expect to observe the
migration of Caloosahatchian taxa to Gatu-
nian waters as temperatures in the Gatunian
approached pre-cooling temperatures of the
Caloosahatchian.
Modern species of Chione are at least par-
tially restricted in their latitudinal distributions
by temperature. No species range beyond
tropical and sub-tropical regions (Fig. 2), and
it can be demonstrated that species’ ranges
have changed in response to changing ther-
mal regimes. For example, the species
Chione undatella (Sowerby, 1835), is abun-
dant in the Upper Pleistocene, interglacial
Millerton Formation of northern California.
The northernmost extent of the species today
is southern California, approximately 640 km
to the south. The modern boundaries of mol-
luscan faunal provinces are frequently asso-
ciated with temperature gradients (Vermeij,
1978), but also with barriers to circulation, for
example Cape Hatteras.
Productivity and Extinction
Unlike the cooling hypothesis, a hypothe-
sis of extinction resulting from declining
planktonic productivity is not based on any
well-documented geological event. Evidence
for higher levels of planktonic productivity in
the tropical Western Atlantic during the
Pliocene is mostly indirect. Petuch (1982)
identified Recent “primary” relict molluscan
faunas of Mio-Pliocene systematic affinities,
off the coast of Venezuela, in cool, upwelling
areas of high planktonic productivity. He in-
ferred that the survival of these communities
in upwelling areas was indicative of the wide-
spread occurrence of these areas in the Mi-
ocene, when the Caribbean was dominated
by such communities. The presence of sys-
tematically related “secondary” relict com-
munities off the Yucatan Peninsula and
Roatan also support this contention (Petuch,
1982). Stanley (1986) hypothesized that a
zone of strong upwelling off the southern ex-
tent of the Florida peninsula during the Early
Pliocene could explain faunal differences be-
tween the Caloosahatchian province and the
nearby Bahamanian fauna, an idea sup-
ported by Cronin’s (1991) ostracod paleo-
temperature data. More recently, Jones &
Allmon (1995) have suggested the occur-
rence of extensive upwelling off the west
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 107
1.0/-0.4
Caloosahatchian
Province
Y Gatunian
Province
FIG. 2. Petuch's (1982) provincial configuration of tropical America during the middle Pliocene. Arrows
indicate that the provinces extend further in those directions. Numbers indicate winter and summer pa-
leotemperature estimates (Cronin, 1991). Map adapted from Jones & Hasson (1985).
coast of Florida during the Early-middle
Pliocene, as evidenced by the ontogenetic
stable isotopic records of various molluscan
taxa. Cronin's (1991) data suggest strongly
the existence of extensive upwelling systems
off the southeast United States and Central
America, during the Pliocene. Cronin (1991)
(see also Raymo et al., 1990) speculated that
during the middle Pliocene, overall global
warmth and reduced amounts of sea ice
resulted in a relatively stronger Gulf Stream
gyre, which in turn caused upwelling along
much of the east coast of North America.
Moreover, given the westward direction of
surficial flow from the Atlantic to the Pacif-
ic, during the Early and middle Pliocene,
through what is now Panama, it is conceiv-
able that extensive upwelling existed in sur-
rounding shelf areas (Stanley, 1986). Allmon
et al. (1993) proposed that declines in pro-
ductivity were responsible for the decimation
of the rich Pliocene molluscan fauna of
southern Florida.
Declining levels of planktonic productivity
would result in different levels of extinction
among different biogeographic regions if the
ecological crisis was more severe in one re-
gion than in another, or altogether absent in
one or more regions. This, for example, would
be the case if the Caribbean in the Early
Pliocene was a region with extensive up-
welling, and has suffered a subsequent de-
cline in the number of upwelling areas and
therefore planktonic productivity (Stanley,
1986; Cronin, 1991). Distinguishing the action
of this agent of extinction from others is dif-
ficult, because there is no reliable, direct
method for assessing levels of productivity in
fossil communities. Changes in planktonic
productivity could, however, affect commu-
nity composition. For example, more produc-
tive habitats may comprise more species than
less productive ones (Brown, 1975; Pianka,
1975), perhaps because higher production
provides more resources for successful utili-
zation by more species (MacArthur, 1965).
108 ROOPNARINE
This type of observation or speculation how-
ever, cannot generally be measured in fossil
communities, because many organisms and
materials (for example, organic detritus) that
form the resource bases of ecological com-
munities have low fossilization potentials
(Dodd & Stanton, 1990).
One possibly relevant parameter that can
be measured easily in the fossil record is
the distribution of body sizes within a com-
munity. Under conditions of declining sus-
pended food supply, a reasonable prediction
is that all suspension-feeding bivalves would
be affected. A further prediction is the dis-
proportionately higher extinction of large
bodied bivalves. In support of this prediction
are observations that: (1) animal body-size
scales negatively with population size (Pe-
ters, 1983), making larger animals more sus-
ceptible to extinction (Vermeij, 1987) under
conditions of ecological duress, (2) the rate of
nutrient intake scales positively with body
size, and (3) conditions of limited resource
supply may lead to communities dominated
by small bodied taxa (Thiel, 1975; Peters,
1983). Vermeij (1978) also points out that
molluscs inhabiting cool, upwelling waters off
Venezuela are significantly larger than con-
specifics elsewhere in the Caribbean. He at-
tributes the differences to higher juvenile
growth rates afforded molluscs in more pro-
ductive waters, but evidence in support of
this remains circumstantial.
This paper presents analyses that demon-
strate that the Chione subgenera collectively
reveal a pattern of greater extinction in the
late Neogene of the Western Atlantic relative
to the Eastern Pacific, coupled with higher
rates of origination in the Eastern Pacific.
Phylogenetic revision of the complex, how-
ever, further demonstrates that the extinc-
tions were not distributed equally among the
subgenera, but seemingly at random. Several
closely related subgenera were affected
more severely than others, while other sub-
genera had higher rates of origination in the
Eastern Pacific.
A summary of the current taxonomic infor-
mation of Chione is presented in the follow-
ing section. A compilation of the geological
histories of the subgenera is also presented.
Hypotheses of extinction caused by cooling
or declining planktonic productivity were
tested by first examining paleobiogeographic
distribution patterns, and then evaluating
body size distributions of late Neogene spe-
cies.
Taxonomic Status of Chione
There are approximately 67 described ex-
tinct and extant species of Chione (although
some of these are undoubtedly synonyms)
(Table 1). The species have been assigned
traditionally, correctly or not, to one of seven
subgenera composing the genus-Chione s.s.,
Chionopsis Olsson, Puberella Fischer-Piette,
Lirophora Conrad, Panchione Olsson, llio-
chione Olsson, Timoclea Brown, and Chion-
ista Keen. While ambiguity of taxonomic ranks
confuses the relationships among subgenera,
this paper will demonstrate that the genus is
paraphyletic. The paraphyletic nature of the
genus can be resolved by revising Chione,
and changing the definition and composition
of the genus.
The above subgenera are all recognized as
members of the subfamily Chioninae Frizzell,
1936, but various authors have argued that at
least some subgenera are different enough
from the definition of Chione (see Palmer,
1927; Olsson, 1961; Keen, 1971, for defini-
tions of Chione) to deserve status as separate
genera. Therefore, Olsson (1961) regarded
Chionopsis as a distinct genus, and both
Woodring (1982) and Ward (1992) treat Liro-
phora similarly. The latter authors also con-
sider Panchione a subgenus of Lirophora,
contrary to Keen (1969). In addition, it is
unclear from a morphological perspective
whether some taxa, such as Chionista, are
members of the Chione clade (as currently
defined), or are nested within other taxa, such
as the related genus Protothaca Dall (Keen,
1971). From a taxonomic viewpoint, the
boundaries between Chione and Protothaca
become obscured when such subgenera as
Chionista and Leukoma Rómer are consid-
ered. These problems underscore the value of
a phylogenetic approach, in which unique
characteristics of taxa are emphasized less,
and relationship among them stressed т-
stead. Recently, Harte (1992a, b) has empha-
sized, using morphological and immunologi-
cal distance data, the close relationship of
Chione to the other chionine genera Merce-
naria Schumacher, and Anomalocardia Schu-
macher. Because of these complications, the
Chione subgenera will be referred to through-
out the rest of the paper collectively as the
Chione complex.
Other Chione subgenera are also problem-
atic from both taxonomic and biogeographic
perspectives. For example, Timoclea, if it
were indeed a subgenus of Chione (see for-
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 109
TABLE 1. List of documented and described species assigned originally to Chione subgenera. Abbreviations
for Localities/Range refer to geological formation, and are explained in Table 3. Asterisked species were
personally examined by the author. Species that have been reassigned to new higher taxa are noted as such
under Comments. Sources consulted frequently were Gardner (1926), Palmer (1927), Grant & Gale (1931),
Parker (1949), Hertlein & Strong (1948), Olsson & Harbison (1953), Olsson (1961, 1964), Perrillat (1963), Jung
(1969), Keen (1971), Abbott (1974), Woodring (1982) and Ward (1992).
_—»”»”»”» жж
Species Age Localities/Range Author(s) Comments
Chione
araneosa* E.—L. EM, BU Olsson, 1942 Described from the Burica
Pliocene Fm. (U. Pliocene) (Olsson,
1942), but also present in
Esmeraldas Fm. (L.
Pliocene)
californiensis* L. Pliocene— Baja Sur, Broderip,
Recent Mexico, L. Pleistocene; 1835
Pt. Mugu,
Calif. —Panama, Recent
cancellata* E. Pliocene— Most shallow Linnaeus,
Recent water deposits in tropical 1767
western Atlantic from m.
Pliocene on.
chipolana* E. Miocene CH Dall, 1903
compta* Recent G. of Calif.—Bayovar, Broderip, 1835
Peru
erosa* m.—L. CA, JB, PB Dall, 1903
Pliocene
guatulcoensis* Recent Pt. Guatulco, Hertlein 8 Strong,
Mexico—Panama Bay 1948
mazyckii* Recent N. Carolina—Cape San Dall, 1902
Roque, Brazil
pailasana E.—m. Venezuela Weisbord, 1964
Pliocene
primigenia E. Pliocene Dominican Rep. Pilsbry & Johnson, = cancellata?
1917
quebradillensis E;,(?) Puerto Rico Maury, 1920 = cancellata?
Pliocene
santodomingensis E. Pliocene Dominican Rep. Pilsbry & Johnson, = cancellata?
1917
undatella* L. Pliocene— SD; $. Calif. — Sowerby, allisoni (Hertlein 8
Recent Paita, Peru, Recent 1835 Grant, 1972) = undatella
Chione
subimbricata* Recent G. of Calif. —Paita, Peru Sowerby, 1835
tumens” L. Pliocene— Baja Calif., Plio.; Verrill, 1870
Recent MZ, Pleist.; Baja Calif.,
Pacific coast and G. of
California
vaca" E.—m. EM, BU Olsson, 1942
Pliocene
Chionista
cortezi* Recent Pacific coast Carpenter,
Baja Calif., and Gulf of 1864
Calif.
fluctifraga* L. Pliocene— Upper Pliocene, Sowerby,
Recent Baja Calif.; s. Calif —Gulf 1853
of Calif.
Chionopsis
amathusia* Recent G. of Calif. — Philippi, 1844
Mancora, Peru
eurylopas M. Miocene BO Woodring, 1982
gnidia* Recent G. of Calif.—Peru Broderip & Sowerby,
1829
jamaniana Pliocene— Pliocene, Pilsbry &
Recent Ecuador; Punta Pasado, Olsson, 1941
Ecuador, Recent
ornatissima Pliocene— Pliocene, Broderip,
Recent Ecuador; 1835
Panama—Ecuador,
Recent
posorjensis L. Oligocene UB Olsson, 1931
procancellata* m.—L. PB, JB, CA Mansfield, formerly Chione
Pliocene 1932
propinqua M. Miocene BO, ZO, DA Speiker, 1922
—E. Pliocene (continued)
110
TABLE 1. (Continued)
Species
rowleei
tegulum*
Chionopsis
walli
woodwardi*
lliochione
subrugosa*
Lirophora
athleta*
alveata*
ballista
carlottae
caroniana
chiriquiensis
clenchi*
dalli
discrepans
ebergenyi
falconensis
hendersoni*
latilirata*
mariae*
obliterata
paphia*
quirosensis
riomaturensis
sellardsi*
tembla
victoria
vrendenburgi
Panchione
burnsii*
funiakensis*
holocyma*
hotelensis
kellettii*
mactropsis*
parkeria
Panchione
trimeris*
ulocyma*
Puberella
bainbridgensis*
cortinaria*
cribraria*
Age
L. Miocene
L. Miocene
Miocene(?)
E.—L.
Pliocene
L. Pliocene—
Recent
E.—L. Pliocene
L. Miocene
E. Oligocene
E. Pliocene
L.(?)
Miocene
E. Pliocene
L. Pleistocene—
Recent
L. Miocene
Recent
E. Pliocene
L. Miocene—
E. Pliocene
L. Pliocene
E. Miocene—
Recent
Recent
Recent
Recent
M. Miocene
. Pliocene
. Miocene
. Miocene
. Oligocene
mrmm
. Miocene
. Miocene
. Miocene
m "mm E
. Miocene—
L. Miocene
L. Miocene
E. Pliocene—
Recent
L. Miocene
E.—M.
Miocene
E. Miocene
m. Pliocene
E. Oligocene
E. Miocene
m.—L.
Pliocene
ROOPNARINE
Localities/Range
GT
GT
Manzanilla,
Trinidad
BW, GB, AX;
Cumana, Venezuela
Baja Sur,
Mexico, L. Pliocene; G. of
Calif —Peru, Recent
Springvale,
Trinidad, W.l.
LI
L. Pleist., Louisiana;
Texas
—G. de Campeche,
Mexico, Recent
ES
Nayarit, Mexico—lslay,
Peru
PN
AL, AN, GT, UR
BW
BE, CA, CV, JB,
PB, WA, YT, ZO; N.
Carolina—Brazil, Recent
G. of Calif. —Guayaquil,
Ecuador
Pacific
Mexico—Panama(?)
West Indies—Brazil
Vicksburg Group, RB, MS,
Mississippi
S
CH
CH
CH, GT;
Chesapeake region
GT
РМ, С. of Сай.—
Реги
АМ, GT
CV, РМ
СН
JB, РВ
Е. Oligocene of
Mississippi, Alabama,
Georgia and Florida
CH; Murfreesboro Stg.,
Virginia
PB, CA; Duplin
Stg., N. Carolina
Author(s)
Olsson, 1922
Brown & Pilsbry,
1911
Guppy, 1866
Guppy, 1866
Wood, 1828
Conrad, 1862
Conrad, 1831
Dall, 1903
Palmer, 1927
Maury, 1925
Olsson, 1922
Pulley, 1952
Olsson, 1914
Sowerby, 1835
Bose, 1906
Hodson, 1927
Dall, 1903
Conrad, 1841
d’Orbigny, 1846
Dall, 1902
Linnaeus, 1767
Hodson, 1927
Maury, 1925
Gardner, 1926
Olsson, 1964
Dall, 1903
Ward, 1992
Dall, 1900
Gardner, 1926
Brown &
Pilsbry, 1911
Olsson, 1922
Hinds, 1845
Conrad, 1855
Glenn, 1904
Gardner, 1926
Dall, 1895
Dall, 1916
Rogers, 1835
Conrad, 1843
Comments
formerly Chione
formerly Chione
numerous
morphs probably representing
different species
formerly Lirophora
formerly Lirophora
formerly Lirophora
formerly Lirophora
formerly
Lirophora; not Mercenaria
(as in Harte, 1992a)
formerly Lirophora
formerly Lirophora
formerly Lirophora
formerly Lirophora
= spenceri, Cooke, 1919;
formerly Chione
formerly Chione
formerly Chione
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 111
TABLE 1. (Continued)
Species Age Localities/Range
intapurpurea* Recent N. Carolina—Brazil
montezuma* Recent Costa Rica—Panama
morsitans* L. Pliocene CA
olssoni Recent Ecuador
pubera L. Miocene L. Miocene,
(?)—Recent Trinidad(?); West
Indies—Brazil, Recent
pulicaria* Recent С. of Calif. —Tumaco,
Colombia
purpurissata Recent G. of Calif —Ecuador
sawkinsi* E.—L. CE, BW
Pliocene
example, Keen, 1971), would be the only sub-
genus to range beyond the Americas, being
found also in the Indo-Pacific and eastern At-
lantic. However, there are several fundamen-
tal morphological differences between Timo-
clea and other members of the Chione
complex; the sculpture is almost entirely ra-
dial, except for irregularly raised points on the
radial lines, which together form an apparent
cancellate pattern. Also, the pallial sinus
tends to be deeper than the normal condition
for Chione, and the hinge plate is not bowed
ventrally, an uncommon condition in Chione.
Similarly, the species C. (Chione) subimbri-
cata (Sowerby) and C. (Chione) tumens (Ver-
rill), were originally classified in the Western
Atlantic and Indo-Pacific genus Anomalocar-
dia (Hertlein & Strong, 1948), but were reclas-
sified in Chione s. s. (Olsson, 1961). Olsson
(1961) argued correctly that C. subimbricata
possesses the hinge characteristics and
some of the sculptural characters of Chione.
Chione tumens is essentially a larger version
of C. subimbricata, and has been considered
by some to be a subspecies of C. subimbri-
cata (for example Keen, 1971). Olsson (1942)
also described a closely related species from
the Lower Pliocene Burica Formation of Pan-
ama, Chione vaca Olsson. Phylogenetic anal-
yses reported later in this paper support
Olsson’s (1961) assignment of these taxa to
Chione s. s., despite the obvious and unique
nature of their sculpture.
Given the obvious taxonomic confusion of
Chione and related genera, it is doubtful that
the examination of diversity patterns and ad-
aptation within the current genus could yield
evolutionarily meaningful results. Such an ex-
amination presumes that the genus is mono-
phyletic, but this presumption cannot be le-
gitimized until a phylogenetic analysis of the
taxa (subgenera) within the genus is under-
taken, and the taxonomic relationships and
Author(s) Comments
Conrad, 1843
Pilsbry & Lowe, 1932
Olsson & Harbison, 1953
Fischer-Piette, 1969
Valenciennes, 1827
formerly Chione
formerly Chionopsis
formerly Chione
Broderip, 1835
Dall, 1902
Woodring, 1925 formerly Chione
character transformations within the clade
established.
Geological History
The Chione complex ranges geologically
from the Early Oligocene (Rupelian Stage)
(Dockery, 1982) to the Recent, and appears
to be restricted to tropical, primarily shallow,
New World waters (Olsson, 1961). The earli-
est occurrences are of Puberella and Liro-
phora in the Lower Oligocene Byram Forma-
tion of Mississippi (Dockery, 1982), and
Puberella in Lower Oligocene strata of An-
tigua (Dockery, 1982). Dall described one of
the Byram species as Chione (Chione) bain-
bridgensis (= spenceri Cooke). The type of
surface sculpture and the depth of the pallial
sinus, however, suggest that this species is
more closely related to Puberella pubera
(Saint-Vincent). Lirophora had extended its
range to the Eastern Pacific by at least the
Late Miocene, occurring in the Zorritos For-
mation of Peru (Woodring, 1982; see Duque-
Caro, 1990, for age of formation). Puberella,
on the other hand, while diverse today in the
Eastern Pacific, has no documented fossil
record in that region.
Chionopsis first occurs in the Late Oli-
gocene (Chattian Stage?) Upper Bohio For-
mation of Panama (Caribbean side) (Wood-
ring, 1982). It was widespread in both the
Eastern Pacific and Western Atlantic during
the Miocene, but is today a paciphilic taxon,
having become extinct in the Western Atlan-
tic by the end of the Pliocene. The earliest
documented occurrence of Chione s.s. is in
the species-rich Lower Miocene (Burdigalian
Stage) Chipola Formation of northwestern
Florida (Chione chipolana Dall, 1903). The
taxon reached the Eastern Pacific by at least
the Early Pliocene (Roopnarine, unpub-
112
TABLE 2. Species used in the phylogenetic analysis. Chione subgenera are listed first. Specimens
ROOPNARINE
belonging to all species, with the exception of Lirophora victoria, were examined by the author.
Genus/subgenus Species Locality/Formation
Chione cancellata Jamaica (Recent)
chipolana Chipola Fm., Florida (Lower Miocene)
Chionisata fluctifraga Gulf of California (Recent)
Chionopsis amathusia Pacific Panama (Recent)
lliochione subrugosa Pacific Panama (Recent)
Lirophora victoria Lower Oligocene (Dockery, 1982)
athleta Caloosahatchee Fm., Florida (Upper Pliocene)
Panchione mactropsis Gatun Fm., Panama (Upper Miocene—Lower Pliocene)
ulocyma Lower Pinecrest Beds, Florida (middle Pliocene)
Puberella cribraria Waccamaw Fm., South Carolina (Upper Pliocene)
Chione tumens Gulf of California (Recent)
Anomalocardia auberiana Florida, Recent
flexuosa Brazil (Recent)
Mercenaria mercenaria South Carolina (Recent)
Protothaca asperrima Pacific Panama (Recent)
Timoclea marica Guam (Recent)
lished), and is today widespread in both the
tropical Western Atlantic and Eastern Pacific.
The Chipola Formation is also the first oc-
currence of Panchione, even though Pan-
chione species have at times been classified
as Lirophora species (Gardner, 1926). Wood-
ring (1982) however, likens the type of
Panchione, P. mactropsis (Conrad) (Late
Miocene, Gatun Formation, Panama), to an-
other species, P. ulocyma (Dall) from the Chi-
pola Formation. It is clear from the descrip-
tion of Panchione (Olsson, 1964) that the
earliest species occur in the Chipola Forma-
tion. Woodring (1982) incorrectly states that
Panchione persists in the Western Atlantic
only until the Late Miocene, for species sim-
ilar (if not identical) to P. ulocyma occur in the
Upper Pliocene Caloosahatchee Formation
of Florida. Panchione is today paciphilic, be-
ing represented by a single Eastern Pacific
species P. kelletii (Hinds) (Woodring, 1982).
The two remaining subgenera, Chionista
and /liochione have brief fossil records. Both
have their earliest documented occurrences
in Upper Pliocene deposits of Baja California
(Durham, 1950). Also, the earliest docu-
mented occurrence of the Chione vaca-sub-
imbricata-tumens species trio is in the Lower
Pliocene Esmeraldas Formation of Ecuador
(C. vaca, personal observation). All three taxa
are today restricted to the Eastern Pacific.
MATERIALS AND METHODS
Phylogenetic Analyses
Twenty-seven morphological characters
were described for species from all the sub-
genera discussed above. Type species were
used whenever specimens were available.
Type species examined include: Chionopsis
amathusia (Philippi), Chione cancellata (Lin-
naeus), Panchione mactropsis (Conrad), /lio-
chione subrugosa (Wood), Chionista flucti-
fraga (Sowerby), Mercenaria mercenaria
(Linnaeus), Anomalocardia flexuosa (Lin-
naeus), and Protothaca (Leukoma) asperrima
(Sowerby). The type species of Timoclea, T.
ovata (Pennant), was not available, so the
Indo-Pacific species T. (Glycydonta) marica
(Linnaeus) was used instead. A complete list
of the species used in the analysis is given in
Table 2. Characters were obtained from both
left and right valves, utilizing the asymmetry
typical of chionine valves, and are discussed
in more detail in Appendix |. The numerically
coded data set is presented in Appendix Il.
Specimens from the following collections
were examined: California Academy of Sci-
ences, Field Museum of Natural History, Flor-
ida Museum of Natural History, Tulane Uni-
versity Geological Collections (the collections
of Drs. Emily and Harold Vokes), University of
California Berkeley (Museum of Paleontol-
ogy), the private collection of Dr. Geerat Ver-
meij, and the author’s own collection.
The data were analyzed, and phylogenetic
hypotheses constructed, using PAUP 3.11
(upgrade of PAUP 3.0, Swofford, 1991). The
branch-and-bound algorithm was used to
provide an exact solution to the search for a
most parsimonious cladogram. All equally
most parsimonious solutions were retained.
Non-binary characters were unordered and
scaled to equal weight based on the number
of states per character (PAUP option
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 113
WEIGHT SCALE; Swofford, 1991). This
method of weighting ensures that characters
with three or more states do not dominate the
resulting trees (Swofford, 1985).
The ingroup comprises the subgenera
Chione, Chionopsis, Puberella, Lirophora,
Panchione, lliochione, Chionista and the
Chione tumens group. The genera Merce-
naria, Anomalocardia, Protothaca and Timo-
clea were treated as outgroup taxa. The out-
group taxa were not constrained to be
outgroups with respect to the ingroup (i.e.,
the cladograms were not rooted with the out-
groups), nor was an a priori hypothesis of
relations among the outgroup taxa included
in the analysis. In fact, preliminary analyses
(Roopnarine, unpublished) — constraining
these genera to be outgroups indicated that
the ingroup cannot be monophyletic with re-
spect to the outgroup taxa. The cladograms
of the present analysis are unrooted.
Optimal character state trees were recon-
structed after analysis according to the
ACCTRAN and DELTRAN criteria. ACCTRAN
maximizes character reversals, and mini-
mizes convergences (Swofford, 1991). This
criterion is therefore a conservative test of
parallel and convergent evolution, which is
suspected for many chionine character
states. DELTRAN forces character transfor-
mations in the opposite direction, favoring
convergence over reversals. A comparison of
the transformations formulated by each algo-
rithm allows a comparison of alternative ev-
olutionary pathways of chionine characters.
Character transformations that are sup-
ported by both algorithms could be consid-
ered particularly robust. Finally, the results of
the analysis were used as a basis to revise
the current taxonomic arrangement of
Chione taxa.
Biogeographic Analysis
The purpose of this analysis was to exam-
ine the geological and geographic distribu-
tions of species within each of the Chione
subgenera, and thereby arrive at conclusions
about subgeneric survival and restriction dur-
ing the late Neogene. The primary focus was
a consideration of differences between the
Eastern Pacific and the Western Atlantic, and
between the Gatunian and Caloosahatchian
provinces. Based on previous work (Wood-
ring, 1966; Vermeij & Petuch, 1986; Stanley,
1986), higher levels of extinction are pre-
dicted to occur in the Western Atlantic com-
pared to the Eastern Pacific. More recent
work (Allmon et al., 1993; Jackson et al.,
1993) suggests that the extinctions in the
Western Atlantic might be matched by spe-
ciation, whereas the Eastern Pacific should
have a higher overall rate of origination.
A list (Table 1) was compiled comprising
all species assigned to the following taxa:
Chione, Chionopsis, Puberella, Lirophora,
Panchione, lliochione, and Chionista. The ge-
nus Anomalocardia was also included, based
on the results of the phylogenetic analyses.
Several species were re-assigned to new
subgenera based on a reconsideration of
subgeneric definitions (Table 1), and some
species names were synonomized on the ba-
sis of the information gathered by specimen
examination and literature descriptions. All
these changes are noted on the species list
for each subgenus (Table 1). Of the 90 spe-
cies listed in Table 1, specimens belonging to
47 of them were examined by this author.
Thirty-nine of the 47 species examined orig-
inated after the Miocene. Literature sources
listed in Table 1 were used to obtain informa-
tion pertaining to the time of first appearance,
and geological and geographical ranges of
each species. A complete listing of geologi-
cal formations considered is given in Table 3,
along with ages, and source of age informa-
tion.
Many Neogene deposits in tropical and
sub-tropical America remain poorly dated,
due to a combination of poor stratigraphic
resolution, the lack of continuous sequences
with index fossils, and discontinuous geolog-
ical study. Several recent advances have
started to resolve the situation, but on prima-
rily regional scales (Duque-Caro, 1990; Jones
et al., 1991; Krantz, 1991; Coates et al., 1992;
Jones, 1995). Therefore, “consensus” ages
were assigned to many of the formations
listed, based on faunal characteristics and
the most recent age estimates available.
The paleobiogeographic data cover the
Early Oligocene to the Recent. In order to
summarize the general biogeographic history
of the subgenera, each chronological epoch
was divided into sub-epochs, according to
Harland et al. (1990). Chronological stages
were not used because that resolution is sim-
ply not yet available for many formations. In
each sub-epoch interval, the number of spe-
cies in each subgenus was listed, along with
the geographic locations or range of the spe-
cies (Table 1). Each location or range was
assigned to one or both of two general re-
gions: the tropical Western Atlantic or East-
ern Pacific. Central American deposits and
the species therein which pre-date Isthmian
uplift (middle Pliocene) were considered to
114
ROOPNARINE
TABLE 3. List of all geological deposits and formations considered in this study. Abbreviations are used
throughout the text. References were used as sources of most current age assignments or
reevaluations.
Formation Abbreviation Age
Aguequexite AX middle Pliocene
Alhajuehla AL Late Miocene
Angostura AN Late Miocene
Bermont BM Early Pleistocene
Bowden BW Late Pliocene
Burica BU middle Pliocene
Byram BY Early Oligocene
Caloosahatchee CA Late Pliocene
Calvert CV E.—Middle Miocene
Cercado CE Early —middle Pliocene
Chipola CH Early Miocene
Culebra CU Middle Miocene
Daule DA L. Miocene—E. Pliocene
Duplin DU Early—middle Pliocene
Eastover ES Late Miocene
Esmeraldas EM Early Pliocene
Gatun GT Late Miocene
Gurabo GB Early—middle Pliocene
Jackson Bluff JB middle Pliocene
La Boca BO Middle Miocene
Limon Group LI middle—Late Pliocene
lower Pinecrest PB middle Pliocene
Beds
Matura MA Early Pliocene
Mint Spring MS Early Oligocene
Montezuma MZ Early Pleistocene
Murfreesboro Early Miocene
Stage
Penita PN Early—middle Pliocene
Red Bluff RB Early Oligocene
Rio Banano RN Early—middle Pliocene
San Diego SD Late Pliocene
Silex Beds SI Early Oligocene
St. Mary's SM Late Miocene
Upper Bohio UB Late Oligocene
Upper Pinecrest PB Late Pliocene
Beds
Urumaco UR M(?)—L(?) Miocene
Waccamaw WA Late Pliocene
Yorktown YT Early —middle Pliocene
Zorritos ZO Late Miocene
Location
Atlantic Mexico
Pacific Panama
Ecuador
Florida
Jamaica
Pacific Panama
Mississippi
Florida
Maryland
Dominican Republic
Florida
Atlantic Panama
Ecuador
South Carolina
Virginia
Ecuador
Atlantic Panama
Dominican Republic
Florida
Pacific Panama
Atlantic Costa Rica
Florida
Trinidad, W. 1.
Mississippi
Pacific Costa Rica
Maryland
Pacific Panama
Mississippi
Costa Rica
California
Florida
Virginia
Pacific Panama
Florida
Venezuela
South Carolina
Virginia
Peru
Reference
Jackson et al., 1993
Woodring, 1982
Duque-Caro, 1990
Lyons, 1991
Stanley, 1986
Coates et al., 1992
Dockery, 1982
Lyons, 1991
Ward, 1992
Saunders et al., 1986
Bryant et al., 1992
Duque-Caro, 1990
Duque-Caro, 1990
Krantz, 1991
Ward, 1992
Duque-Caro, 1990
Coates et al., 1992
Saunders et al., 1986
Lyons, 1991
Woodring, 1982
Coates et al., 1992
Jones et al., 1991
Jung, 1969
Dockery, 1982
Coates et al., 1992
Ward, 1992
Coates et al., 1992
Dockery, 1982
Coates et al., 1992
Hertlein 8 Grant, 1972
Ward, 1992
Woodring, 1982
Jones et al., 1991
Krantz, 1991
Krantz, 1991
Duque-Caro, 1990
belong to a region based on characteristics
of faunal composition and whether they are
located on the Pacific or Atlantic sides of the
isthmus. Contemporaneous Neogene depos-
its on opposite sides of the isthmus are often
very different faunistically (Duque-Caro,
1990; Coates et al., 1992), possibly reflecting
the action of oceanographic barriers, the age
of initial isthmus uplift, and differences in tec-
tonic and sedimentary histories. The diversity
within each region was then documented by
summing the number of species in each of
the two regions for successive sub-epoch in-
tervals. Levels of speciation and extinction
were assessed as the numbers of first and
last occurrences per region per sub-epoch.
Note that by adopting this approach, extinc-
tions can only be constrained to the sub-ep-
och following a last occurrence. Biostrati-
graphic discontinuity for a subgenus does
not necessarily represent true extinction, but
is also dependent on stratigraphic continuity,
which as already noted is problematic for
much of Neogene tropical America.
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES VAS
Testing the hypothesis that cooling caused
the late Neogene extinctions requires a fine-
scale latitudinal resolution of biogeographic
distributions. Due both to the absence of
widespread, stratigraphically continuous late
Neogene sections in tropical America, and
the coupled uncertainties in paleobiogeo-
graphic boundaries, latitudinal resolution is
limited. Using Petuch’s (1982) scheme, | have
divided the entire region into three areas (Fig.
2): the tropical Pacific Gatunian Province,
which was roughly equivalent to the Recent
Panamic Pacific region; the Atlantic Gatunian
Province, which encompassed the modern
day Caribbean Sea; and the Caloosahatchee
Province, which extended from the Florida
peninsula to South Carolina. The Florida pen-
insula during the Pliocene may have repre-
sented a zone of transition between the trop-
ics and sub-tropics (Stanley, 1986). Species
from Florida were assigned, at least initially,
to the Caloosahatchian category, a decision
supported in Roopnarine (1995).
The last occurrences of species within
each subgenus were placed within this
framework for the Early-middle (5.2-2.5 mya)
and latest Pliocene (2.4-1.6 mya). A general
analysis of extinction levels was performed
by summing the number of species within
each province during each sub-epoch. The
analysis was also performed using time inter-
vals based on the latest age estimates of rel-
evant deposits. A hypothesis of cooling, as
formulated by Stanley (1984), cannot be re-
jected if a higher level of extinction is noted in
the tropical Atlantic Gatunian Province com-
pared to the subtropical Caloosahatchian
Province, after 2.4 Ma.
Body Size
As noted earlier, declining levels of plank-
tonic productivity in the late Neogene tropical
Western Atlantic has been cited as a proxi-
mal cause of the extinctions. Changing the
nutrient supply to a community should alter
the trophic composition of the community.
Hypothetically, larger-bodied species are af-
fected more adversely than smaller, trophi-
cally equivalent (all suspension-feeders) spe-
cies. This statement is based on the following
observations: large-bodied species, while
numerically inferior with respect to smaller
species, nevertheless account for compara-
ble quantities of biomass (Stanton & Nelson,
1980; Stanton et al., 1981; Staff et al., 1985);
and while smaller poikilotherms ingest rela-
tively larger quantities of food than do larger
ones (the amount of food ingested by poiki-
lotherms is roughly four times their metabolic
rates), the rate of nutrient intake scales pos-
itively with body size (Peters, 1983).
[=078WS==
where | = ingestion rate and W = body weight
(Peters, 1983).
In order to test a hypothesis of declining
levels of planktonic primary production, max-
imum body size was documented for as
many Atlantic Pliocene, and Atlantic and Pa-
cific post-Pliocene chionine species as were
available. Body size was defined simply as
maximum valve height, and was measured
with digital calipers to the nearest 0.01 mm.
The number of specimens examined for each
species was noted (see Results, Table 4), to
caution that this type of analysis is prone to
sample-size bias. Articulated valves were
counted as single individuals. Species col-
lections with many specimens may have in-
creased chances of containing very large
specimens (due purely to sampling bias),
analogous to the rarefaction relationship be-
tween species richness and sample size ob-
served in ecological studies (Sanders, 1968).
This potential source of bias is compensated
for, however, by the observation that small-
bodied species are more abundant in depos-
its than larger species, are therefore generally
represented by larger samples, and hence
have an increased probability of containing
specimens near the maximum size of the
population from which the sample was de-
rived. Empirical rarefaction curves could not
be used in this instance to verify the obser-
vation quantitatively, because the derived
curves would not be independent of time
(Raup, 1975).
The maximum body size of a species was
defined as the body size of the largest spec-
imen measured. In cases when a species
was not represented adequately in one of the
museum or author’s collection, maximum
size was taken from a literature description of
the species, primarily from Abbott (1974),
Keen (1971) and Palmer (1927).
Many fossil species are known from single
localities or very restricted geographic areas.
A review of Table 1, however, will show that
many extant species have very large geo-
graphic ranges, and therefore undoubtedly
exhibit ecologically based variation. There-
fore, when documenting body size for Recent
116 ROOPNARINE
Chione chipolana
Mercenaria mercenaria
Puberella cribraria
Timoclea marica
Protothaca asperrima
Chionista fluctifraga
Chione tumens
Chione cancellata
Chione chipolana
Iliochione subrugosa
Anomalocardia flexuosa
Anomalocardia auberiana
Lirophora athleta
Lirophora victoria
Panchione mactropsis
Panchione ulocyma
FIG. 3. Cladograms resulting from analysis of Chione shell characters. Incongruency among the cla-
dograms is entirely dependent upon the placement of /liochione.
species, geographic range and location were
considered.
RESULTS
Phylogenetic Analyses
Analysis of the character data by PAUP re-
sulted in three equally parsimonious cla-
dograms (Fig. 3), each 106 steps in length,
and with consistency indices of 0.552, ho-
moplasy indices of 0.448 and retention indi-
ces of 0.602. The cladograms are similar in
topology, differing only in the placement of
the species /liochione subrugosa. The spe-
cies lliochione subrugosa and Lirophora ath-
leta (Conrad) have zero branch lengths. They
could thus be identified as potential ances-
tors, or as possessing the same combination
of character states as an ancestor. The zero
branch length for lliochione subrugosa re-
sults, however, from the exclusion of charac-
ter 426 (radial indentation of posterior valve
surface), which is autapomorphic in /. subru-
gosa, from the analysis. The character was
included in the data matrix because it may
become informative if more characters or
taxa are added at a later time, or if the data
set is later analyzed at a higher level of uni-
versality (Wiley, 1981; S. Carlson, personal
communication). It also indicates that /. sub-
rugosa possesses ancestral states for many
characters, but is most likely not an ancestral
taxon. Lirophora athleta has a zero branch
length, possibly because its sister species L.
victoria (Dall) has been coded with several
missing character states. Lirophora victoria is
the earliest documented species of Lirophora
(Early Oligocene), and not all character states
could be coded with confidence. The results
of the analysis could therefore be interpreted
to indicate L. athleta as a potential ancestor.
No autapomorphies could be identified to
distinguish the two species, reflecting a com-
mon problem when dealing with morpholog-
ical “species” of Lirophora (see, for example,
Ward, 1992). All three cladograms identify
species in the same subgenus as sister spe-
cies: Chione cancellata and C. chipolana;
Lirophora athleta and L. victoria; Panchione
ulocyma and P. mactropsis; and Anomalo-
cardia auberiana and A. flexuosa.
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES ul
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Timoclea marica
Protothaca aspirrima
Chionista fluctifraga
Chione tumens
Chione cancellata
Chione chipolana
Iliochione subrugosa
Anomalocardia flexuosa
Anomalocardia auberiana
Lirophora athleta
Lirophora victoria
Panchione mactropsis
Panchione ulocyma
FIG. 4. Strict consensus tree of cladograms illustrated in Fig. 3.
Strict and 50% majority rule consensus
trees were used to summarize the informa-
tion of the three cladograms (Fig. 4, 5). Both
trees are well resolved and identical in topol-
ogy because of the high congruency among
the source cladograms. The incongruencies
among the cladograms result in a polytomy
from which branch Anomalocardia, Шо-
chione, and the sister taxa Lirophora and
Panchione. The ingroup is divided into two
sister clades containing the following spe-
cies: first, Chionista fluctifraga, Chione tu-
mens, C. cancellata and C. chipolana; and
secondly, Anomalocardia auberiana, A. flex-
uosa, Iliochione subrugosa, Lirophora
atheleta, L. victoria, Panchione mactropsis,
and P. ulocyma. The inclusion of Anomalo-
cardia in the large Chione clade, and the sur-
prising exclusion of Chionopsis amathusia
and Puberella cribraria, indicate that Chione,
as currently defined, is a paraphyletic genus.
The branching position of Timoclea confirms
its status outside the main clade of Chione
subgenera. The structure of the cladograms
and the consensus trees suggest strongly the
need for a thorough taxonomic revision of
Chione. Revising the taxonomy of Chione on
the basis of a phylogenetic analysis pre-
sumes that the phylogenetic hypotheses are
more informative about the interrelationships
of the Chione subgenera than is the tradi-
tional taxonomy.
118
ROOPNARINE
100
100
100 100
00
100
100
100
100
100
100
100
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Timoclea marica
Protothaca aspirrima
Chionista fluctifraga
Chione tumens
Chione cancellata
Chione chipolana
Iliochione subrugosa
Anomalocardia flexuosa
Anomalocardia auberiana
Lirophora athleta
Lirophora victoria
Panchione mactropsis
Panchione ulocyma
FIG. 5. 50% majority rule consensus tree of cladograms illustrated in Figure 3. This tree is identical in
topology to the strict consensus tree (Fig. 4).
Character Optimization and Evolution
The three cladograms are very similar in
topology, but the ACCTRAN and DELTRAN
reconstructions differ for several significant
characters. Perhaps a useful manner in which
to view the data will be to examine the sup-
port for the three major clades that are a con-
sistent feature of all the cladograms; the
overall Chione clade (including Anomalocar-
dia, but excluding Chionopsis and Puberella),
the Chione-Chionista clade (hereafter Chione
subclade) and the Lirophora-Panchione-
Anomalocardia-lliochione clade (hereafter Li-
rophora subclade). Character transforma-
tions supported by several cladograms can
be regarded as robust, but unresolved differ-
ences have to await the addition of more
character information to the analysis. The fol-
lowing character transformations are all illus-
trated on cladogram 1. Interior nodes are la-
belled on Figure 6, and the synapomorphies
uniting taxa are listed in Appendix 11.
Chione Clade
Two characters are apomorphic at the an-
cestral node of the Chione clade on all cla-
dograms, and support the monophyly of the
clade. Two additional characters are apo-
morphic on the basis of ACCTRAN and DEL-
TRAN reconstructions respectively.
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 119
28
26
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Timoclea marica
—_— 27
29 | |—— Protothaca asperrima
Chionista fluctifraga
Chione tumens
18
— Chione cancellata
17
Chione chipolana
Iliochione subrugosa
ь Anomalocardia flexuosa
Pl ИХ)
Anomalocardia auberiana
Lirophora athleta
20
| 2 == Lirophora victoria
== Panchione mactropsis
21
———— Panchione ulocyma
FIG. 6. Character state tree of Character #1 (depth of pallial sinus). Key to character states: (0) solid
line—reduced; (1) double line—short; (2) double line, upper (left) thick—deep; (3) double line, lower (right)
thick—very deep. Transformations at labelled interior nodes are listed in Appendix III.
Character #20 (condition of right middle
cardinal tooth) (Cl = 0.667) is apomorphic at
the ancestral node on all three cladograms
(node 26) and is reconstructed identically by
the ACCTRAN and DELTRAN algorithms.
The character is mapped onto cladogram 1
to illustrate the changes (Fig. 6). The plesio-
morphic state is a tooth with a shallow, surf-
icial groove. At node 26, the tooth becomes
smooth. The only exception is Chionista fluc-
tifraga, which evolves a bifid tooth. This con-
dition is apparently convergent with the bifid
tooth of Mercenaria.
Character +7 (distal edge of concentric
sculptural lamellae) (Cl = 0.500) is apomor-
phic at node 26 on all three cladograms, but
is reconstructed differently by ACCTRAN and
DELTRAN. Based on the ACCTRAN recon-
struction (Fig. 7), the plesiomorphic state is a
concentric sculptural element with a sharp
distal edge. At the Chione ancestral node, the
sculptural edges become smoother, but re-
vert (converge in the DELTRAN reconstruc-
tion) to a sharp morphology at the ancestral
node of the Chione subclade. Both Chione
chipolana and Puberella cribraria have sculp-
tural elements with reinforcing ridges on the
distal edges. This similarity is indicated to be
a homoplasy.
Character #8 (orientation of concentric
120 ROOPNARINE
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Timoclea marica
Protothaca asperrima
mas Chionista fluctifraga
о Anomalocardia flexuosa
'—— Anomalocardia auberiana
ne athleta
me victoria
Chione tumens
Chione cancellata
Chione chipolana
Tliochione subrugosa
= Panchione mactropsis
——— Panchione ulocyma
FIG. 7. Character #7 (summit of concentric sculpture). Key (lines same as for Fig. 6): (0)—sharp; (1)—
smooth; (2) —summit reinforced.
sculptural elements) (Cl = 0.667) is apomor-
phic at the Chione clade's ancestral node
(Fig. 8), but is reconstructed differently by the
ACCTRAN and DELTRAN algorithms. The
plesiomorphic state is vertical sculpture,
which reappears in the Chione (Chione) sub-
clade. The apomorphic state is folded sculp-
ture which is flattened in the dorsal direction.
This type of concentric sculpture is typical of
the Lirophora subclade, and is useful in de-
fining the group. Chione chipolana, however,
also has sculpture that appears to be partially
folded. The sculpture is vertical at its base,
but is foliaceous and becomes folded toward
the summit. This variation may be synapo-
morphic with the ancestral sculpture of the
Lirophora subclade, but such a hypothesis
cannot be demonstrated with the material
currently available. The rounded nature of the
sculpture in Chione tumens is shared only by
C. vaca and C. subimbricata (not included in
this study).
Lirophora Subclade
There are four characters that strongly
support the Lirophora subclade; #1, depth of
pallial sinus, CI = 0.600; #2, type(s) of sculp-
ture on valve surface, CI = 0.800; #8, orien-
tation of concentric sculptural lamellae, CI =
0.667 (discussed above); #11, morphology of
nymph, Cl = 0.500. All these were recon-
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 121
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Timoclea marica
Protothaca aspirrima
nés Chionista fluctifraga
—
Chione tumens
Chione cancellata
Chione chipolana
Iliochione subrugosa
m Anomalocardia flexuosa
= Anomalocardia auberiana
Ml Lirophora athleta
Lirophora victoria
== Panchione mactropsis
|
——— Panchione ulocyma
FIG. 8. Character #8 (orientation of concentric sculpture). Key (as in previous figures): (0)—vertical; (1)—
folded dorso-ventrally; (2)—rounded.
structed identically by ACCTRAN and DEL-
TRAN (Figs. 9, 10), with the exception of
character #8. Character #8 was already dis-
cussed as being apomorphic at the ancestral
node of the overall Chione clade, but can be
used to distinguish between the Lirophora
subclade and the Chione subclade.
The pallial sinus is generally reduced in all
chionine taxa, compared to other venerid
subfamilies. It is further reduced in the Liro-
phora subclade, being present but extremely
small. This is probably a reflection of the
shallow burial of these clams during life. The
pallial sinus is completely absent in Anoma-
locardia flexuosa, a species used in this anal-
ysis, but is present in other species assigned
to Anomalocardia, for example A. auberiana.
The state in these other species is identical to
the state in the other taxa of the Lirophora
subclade. The presence of much reduced or
absent pallial sinuses in the Chione subclade
is convergent with the states in the Lirophora
subclade.
Perhaps the character most diagnostic of
the Lirophora subclade is the morphology of
the nymph. This is a binary character, the
nymph being either smooth, or roughened, a
condition described as “rugose.” All taxa
nested within the Lirophora subclade have
rugose nymphs. The only other taxon with a
rugose nymph is Mercenaria, but this is ap-
parently a convergent condition.
122 ROOPNARINE
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
Г. Timoclea marica
Protothaca asperrima
AT er Chionista fluctifraga
Chione tumens
——— Chione cancellata
ES Chione chipolana
Iliochione subrugosa
Anomalocardia flexuosa
ms Anomalocardia auberiana
Lirophora athleta
| Lirophora victoria
| = Panchione mactropsis
~~ Panchione ulocyma
FIG. 9. Character #2 (type of sculpture present). Key (as in previous figures): (0)—concentric only; (1)—radial
and concentric, concentric dominant; (2) both, radial sub-obsolete; (3) triple line—both sub-obsolete; (4)
double line, upper thick—both, radial dominant.
Like nymph morphology, the morphology
of the sculptural elements is diagnostic of the
Lirophora subclade. Taxa comprising the Li-
rophora subclade have both radial and con-
centric sculptural elements, but the radial el-
ements tend to be faint to obsolete.
Lirophora itself has no radial elements
present, but this can be viewed as a com-
plete loss of radial sculpture, the concentric
sculpture being synapomorphic with the rest
of the subclade. The absence of radial sculp-
ture in both Lirophora and Mercenaria should
therefore be recognized as homoplastic,
both states being the result of the loss of a
character.
Chione Subclade
The Chione subclade is supported strongly
by at least three characters, #1, #8 and #10
(anterior cardinal tooth of left valve, Cl =
0.500) (Fig. 11), two of which (1 and 10) are
reconstructed identically by ACCTRAN and
DELTRAN. The pallial sinus is altogether ab-
sent in all species of Chione s.s., and Chion-
ista, with the exception of Chione chipolana.
Chione chipolana’s pallial sinus appears to
be convergent with the state in the Lirophora
subclade. The plesiomorphic condition of
character #10 is a relatively wide tooth, but
the tooth is noticeably narrow in both the
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 123
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
'— Protothaca asperrima
| Timoclea marica
+i pee Chionista fluctifraga
Chione tumens
—— Chione cancellata
ae Chione chipolana
Iliochione subrugosa
Anomalocardia flexuosa
Anomalocardia auberiana
Lirophora athleta
Lirophora victoria
Panchione mactropsis
Panchione ulocyma
FIG. 10. Character #11 (condition of nymph). Key as in previous figures. (0)—rugose; (1)—smooth.
Chione subclade and Mercenaria (the result
of convergence).
The orientation of the concentric lamellae
is vertical in Chione s.s., and this is probably
the retention of a plesiomorphic character
state (contrary to the ACCTRAN reconstruc-
tion). Chione tumens has a truly unique type
of sculpture, and it is unclear how it is derived
from any of the character states in the Chion-
inae.
Biogeographic Analyses
All the Chione subgenera (with the excep-
tion of Chionista) first appear in the Western
Atlantic. The number of species described
from the Early Oligocene through the Middle
Miocene (35.4-16.3 Ma) is low (Fig. 12). The
Early Miocene is an exception, but most of
the species recorded here (85.7%) are from
the very rich Chipola Formation of northwest
Florida. Woodring (1982) suggested that
some Panchione species documented there
may be synonyms and require revision. Three
of the four Chipola Panchione species how-
ever were examined by this author, and all
are consistently recognizable. It should also
be noted that the number of well-described
Oligocene and Lower Miocene soft-sediment
deposits in tropical America is relatively
small, and accurate chronological and bio-
stratigraphic dating is problematic. The num-
124 ROOPNARINE
Mercenaria mercenaria
Puberella cribraria
Chionopsis amathusia
fe Timoclea marica
Protothaca asperrima
ro Chionista fluctifraga
Chione tumens
Chione cancellata
Chione chipolana
[liochione subrugosa
Anomalocardia flexuosa
Anomalocardia auberiana
Lirophora athleta
Lirophora victoria
= Panchione mactropsis
——— Panchione ulocyma
FIG. 11. Character #10 (condition of left valve anterior cardinal tooth). Key as in previous figures. (0)—tooth
wide; (1) —tooth narrow.
ber of species reported here for this period is
therefore probably not a dependable reflec-
tion of actual diversity.
There is a steady increase in diversity from
the Middle Miocene to the Early Pliocene
(16.3-5.2 Ma) in both the Western Atlantic
and Eastern Pacific (Fig. 12), corresponding
in part to an increase in the number of de-
posits available for sampling. During this pe-
riod, increasing diversity in the Western At-
lantic is accounted for primarily by the
appearance of new species, which is accom-
panied by an increasing level of last appear-
ances (Fig. 12). In the Eastern Pacific on the
other hand, increasing diversity is the result
of first appearances, coupled with a low rate
of extinction, and therefore higher species
longevities.
The level of extinction in the Western At-
lantic continues to increase into the Late
Pliocene, and the proportion of species that
last appear in the Late Pliocene greatly ex-
ceeds the number of first appearances (Fig.
13). The result is a dramatically lower diver-
sity in the Recent. In contrast, the rate of ex-
tinction in the Eastern Pacific decreases dur-
ing the late Neogene (Fig. 13). The proportion
of new species, though also decreasing, is
never exceeded by the proportion of species
going extinct. There are many species (17)
which first appear in the Eastern Pacific dur-
ing the Pleistocene and Recent; this results in
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 129
No. of species
25
20
15
10
Sub-epoch divisions
FIG. 12. Total species diversity of Chione subgenera since the Early Oligocene. Chronological (horizontal
axis) intervals are geological sub-epochs. Graphs on upper left represents sum diversity of traditional
subgenera. Upper right—Chione clade (as defined in this paper); lower left—Chionopsis; lower right—
Puberella. Circles—total number of species present; squares—number of first appearances; triangles—
number of last appearances.
a higher diversity than in the Western Atlan-
tic. The Pliocene is also the time of first ap-
pearance of the strictly Eastern Pacific sub-
genera, Chionista, lliochione, and the Chione
vaca-subimbricata-tumens trio.
In summary, these data agree well with ob-
servations that the Western Atlantic suffers
heavier extinction during the Pliocene (82.6%)
than does the Eastern Pacific (38.5%) (Wood-
ring, 1966; Stanley, 1986; Vermeij & Petuch,
1986) (Fig. 13). The Eastern Pacific exhibits a
higher rate of origination during the post-
Pliocene compared to the Western Atlantic,
resulting in a higher diversity in the Eastern
Pacific (17 vs. 7 new species). The overall
pattern is reflected by the individual subgen-
era. All exhibit higher levels of extinction in the
Western Atlantic relative to the Eastern Pa-
cific, and higher levels of origination in the
Eastern Pacific during the post-Pliocene. Ex-
tinction within these Chione subgenera in the
Western Atlantic was not matched by speci-
ation.
These results cover the entire Pliocene
though, obscuring the relative timing of the
disappearances, and hence the action of an
extinction agent such as cooling. In order to
focus on the time of the extinctions, it be-
comes necessary to assign estimated ages
to sampled deposits. Given the contentious
nature of aging Neogene tropical American
deposits, the most current age estimates
available were relied upon, coming primarily
from the following references; Coates et al.
(1992), Duque-Caro (1990), Jones et al.
(1991) and Krantz (1991). These works either
126 ROOPNARINE
82.6% Pliocene
„20
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”
510
ö
Z 5
0
AS G
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S à
(à
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25 post-Pliocene
66.7%
„20
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015
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ö
25
0
LO ©
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FIG. 13. Late Neogene changes in diversity. Upper
graph illustrates levels of extinction during the
Pliocene in the western Atlantic and the eastern
Pacific. Shaded areas represent number of extinct
species, unshaded represent survivors. Lower
graph illustrates levels of origination in the Pleis-
tocene and Recent. Shaded areas represent new
species.
re-evaluate and assign ages, or summarize
current estimates. The Early Pliocene (5.2-
3.4 Ma) of the Atlantic Gatunian is covered
in this set of data by species from the
Agueguexquite Formation of Mexico (Perril-
lat, 1963), the Cercado and Gurabo deposits
of the Dominican Republic (Saunders et al.,
1986), the Rio Banano Formation of the
Limon Group in Costa Rica (Coates et al.,
1992) and the Tubara Formation of Colombia
(Duque-Caro, 1990). All these formations
probably pre-date the initiation of Northern
Hemisphere cooling (—2.4 Ma), or end shortly
thereafter. They could therefore be listed as
Early-‘‘middle’’ Pliocene. The Lower Pliocene
deposits of the Caloosahatchian Province,
namely the Jackson Bluff Formation, the
lower Pinecrest Beds, the Duplin Formation,
the Raysor Formation and the Yorktown For-
mation, all range past the official stage
boundary of the Late Pliocene (Jones et
al., 1991; Krantz, 1991), 3.4 Ma, but termi-
nate about 2.5 Ma. Therefore they too could
be considered technically as Early-middle
Pliocene. The Atlantic Gatunian Bowden For-
mation is Late Pliocene in age (Stanley,
1986), as are the Caloosahatchian Caloosa-
hatchee Formation, upper Pinecrest Beds,
the Murfreesboro Stage (Ward, 1992) and the
Waccamaw Formation (Lyons, 1991; Jones
et al., 1991). These formations are all < 2.5
million years old. Early-middle Pliocene East-
ern Pacific deposits comprise formations
such as the Burica, Esmeraldas and Pro-
greso formations, while the Late Pliocene is
represented primarily by deposits from Baja
and southern California.
Placed in a temporal framework of Early-
middle and Late (latest) Pliocene categories,
the pattern of extinction is striking. Only 50%
of all species in the Early-middle Pliocene of
the Western Atlantic were extinct by the Late
Pliocene, but 73.3% of Late Pliocene species
are absent from the Pleistocene (Fig. 14). On
the other hand, extinction levels in the East-
ern Pacific are 57.1% and 11.1% respec-
tively for the Early-middle and Late Pliocene.
The extinctions would therefore seem to be
concentrated in the Late Pliocene of the
Western Atlantic. This result is consistent
with the compilations of Allmon et al. (1993)
and Jackson et al. (1993). At the subgeneric
level however, a different pattern emerges
(Fig. 15). In the Western Atlantic, the subgen-
era Lirophora and Panchione experience
much higher levels of extinction (60% and
100% respectively) during the Early-middle
Pliocene, than in the Late Pliocene. The sub-
genera Chione, Chionopsis and Puberella do
not exhibit heightened levels of extinction un-
til the Late Pliocene.
The difference of timing among the sub-
genera is not explained easily. The data
could be biased biogeographically if the sub-
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 127
Atlantic Pacific
FIG. 14. Timing of Pliocene extinctions in Western
Atlantic and Eastern Pacific. In each category, bar
on the left summarizes data for the Early-middle
Pliocene, bar on right the Late Pliocene. See text
for explanation of chronological intervals. Percent-
ages refer to percent of diversity extinct in next
chronological interval. Shading as in Fig. 13.
genera were not distributed evenly between
the Atlantic Gatunian region and the
Caloosahatchian Province, and if one of the
two regions experienced more severe extinc-
tion. The extinctions are probably not related
to phylogenetic history. It is interesting to
note that Lirophora and Panchione occupy a
separate subclade that could have been dec-
imated by a selective extinction agent (Fig. 4).
If the extinction was biased against Lirophora
and Panchione however, then Anomalocardia
should exhibit the same pattern. It does not,
and it seems therefore that any hypothesis of
phylogenetic selectivity of the extinction can
be rejected.
In order to examine the role of biogeo-
graphic distribution in the extinction, it is nec-
essary to assign species to one or both of
two biogeographic regions, the Atlantic Ga-
tunian region and the Caloosahatchian Prov-
ince. Interestingly, all Pliocene species of
Chione were endemic to one of the two re-
gions, with the possible exception of Chione
cancellata. Even this species, however, may
in fact be two separate taxa (Roopnarine,
1995). Species can therefore be placed easily
into one or two of the following categories:
Early-middle Pliocene Atlantic Gatunian;
Early-middle Pliocene Caloosahatchian; Late
Pliocene Atlantic Gatunian; Late Pliocene
Caloosahatchian. The results indicate that
extinction of Early-middle Pliocene species in
the Atlantic Gatunian region was 53.8%,
while in the Caloosahatchian Province it was
only 18.2% (Fig. 16). By the latest Pliocene,
however, extinction declined slightly in the
Atlantic Gatunian to 50%, but climbed to
58.3% in the Caloosahatchian.
The high level of extinction in the Atlantic
Gatunian during the Early-middle Pliocene is
due almost entirely to the extinction of spe-
cies assigned to Lirophora and Panchione
(Fig. 17). Both subgenera also experience
higher levels of extinction at this time, in the
Caloosahatchian Province, relative to other
subgenera. Furthermore, it is apparent that
Lirophora, with its numerical dominance of
the species diversity of both the Atlantic Ga-
tunian and Caloosahatchian, contributes the
most to the extinction. During the latest
Pliocene all surviving subgenera experience
high levels of extinction in both geographic
regions, with the exception of Anomalocar-
dia.
Body Size
Early-middle Pliocene species of the
Caloosahatchian Province all exceed 35 mm
in height (Table 4). The largest species is
Chionopsis procancellata (Mansfield) from the
lower Pinecrest Beds, the largest specimen of
which measured 57.75 mm. Lirophora athleta
from the lower Pinecrest Beds reached a
height of at least 35.5 mm. Species from the
same deposit include Chione erosa, which
reached a height of 47.35 mm, and Panchione
ulocyma, which reached a height of 43.30 mm.
Puberella cortinaria (Rogers) from the Jack-
son Bluff Formation was measured at 37.03
mm. Species from the northern regions of the
Atlantic Gatunian Province tend to be much
smaller. There are no records of Early-middle
Pliocene specimens of Chionopsis wood-
wardi (Guppy) or Puberella sawkinsi (Wood-
ring) from the Cercado or Gurabo Forma-
tions exceeding 30 mm in height, nor of
specimens of Chione primigenia Pilsbry &
Johnson or C. santodomingensis Pilsbry &
Johnson (Palmer, 1927). Interestingly, there is
some evidence that species from the south-
ern portion of the province, for example the
Rio Banano Formation of Costa Rica, were
larger. Woodring (1982) recorded a specimen
of Panchione mactropsis of 51.5 mm height
from the Rio Banano. Chionopsis tegulum
(Brown & Pilsbry), while not measured for this
study, is a large species, attaining heights in
128 ROOPNARINE
6
5
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S.
n 3 Y
= Y
Ро й
о A À
=. 7
0 © © > |
ERS
ee
< 0 © © A (© ©
[в © ve
ys
in Fig. 14.
FIG. 16. Temporal and biogeographic categoriza-
tion of Pliocene extinctions in the western Atlantic.
Shading represents extinct species. Abbreviations:
E.-m.—Early and middle Pliocene; L.—Late
Pliocene; Gatunian—Atlantic Gatunian region;
Caloosa.—Caloosahatchian Province.
excess of 45 mm in the Upper Miocene Ga-
tun Formation. It also persists into the Lower-
middle Pliocene Rio Banano Formation. The
6
E.-m. Pliocene
> a
No. of species
м ©
Г КБУ]А--ЗБЗ]Б-ЪЪЫ Зы
290 À + 2 2
se eS & e oe oO
6
on
>
No. of species
№ w
FIG. 17. Subgeneric breakdown of data presented
in Fig. 16. Per subgenus, bars on right represent
the Caloosahatchian Province, and on left the At-
lantic Gatunian region. Key to bars as in previous
figures.
only Early Pliocene sizes available from the
Eastern Pacific are of the species Chionopsis
amathusia and Chione vaca (Olsson) from the
Esmeraldas Formation of Ecuador. Both ex-
ceeded 30 mm in valve height.
The situation alters significantly in the lat-
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES
129
TABLE 4. Valve heights measured for samples of chionine bivalves. Maximum valve height recorded for
each sample.
иди р
Species
Chione erosa
Chione cancellata
(United States)
Chione cancellata
(West Indies)
Chione mazyckii
Chione undatella
Chionopsis
procancellata
Chionopsis woodwardi
Chionopsis amathusia
Chionopsis gnidia
Lirophora athleta
Lirophora latilirata
Lirophora hendersoni
Lirophora paphia
Panchione ulocyma
Puberella cribraria
Puberella intapurpurea
Puberella morsitans
Puberella sawkinsi
Puberella pulicaria
Chionista cortezi
Chionista fluctifraga
lliochione subrugosa
Chione tumens
Chione raca
Age
E.—middle
Pliocene
E.—middle
Pliocene
E.—middle
E:
L.
E
(ER
E:
M. Pleistocene
E
Pliocene
Pliocene
Pliocene
Pliocene
Pliocene
Pleistocene
Pleistocene
Recent
Recent
Recent
Recent
Recent
Recent
Recent
Recent
Recent
Recent
Recent
m
. Pliocene
. Pliocene
. Pliocene
. Pliocene
. Pliocene
. Pliocene
Recent
Recent
E.
. Pliocene
. Pliocene
. Pliocene
. Pliocene
. Pliocene
. Pleistocene
mMrrrrm
Pliocene
Holocene
LE
Pliocene
Recent
E.—m. Pliocene
L.
lE
Pliocene
Pliocene
Recent
L.
L;
Pliocene
Pliocene
Recent
Recent
Recent
Recent
Recent
E:
Pliocene
Locality/Formation
Raysor Fm., South Carolina
Pinecrest Beds, Bird Rd., Florida
Pinecrest Beds, Collier Co., Florida
Pinecrest Beds, Sarasota, Florida
Caloosahatchee Fm., Palm, Beach Co.,
Florida
Caloosahatchee Fm., Palm Beach Co.,
Florida
Waccamaw Fm., South Carolina
Bermont Fm., Highlands Co., Florida
Ft. Thompson Fm., Collier Co., Florida
Anastasia Fm., Palm Beach Co., Florida
Sanibel Is., Florida
Palm Beach Co., Florida
South Carolina
Bahamas Islands
Jamaica
St. Thomas, U.S. Virgin Is.
Venezuela
South Carolina
Laguna San Ignacio, Baja California,
Mexico
Bahia San Luis, Baja California, Mexico
San Carlos, Baja California, Mexico
Lower Pinecrest Beds, Sarasota Co.,
Florida
Highlands Co., Florida
Palm Beach Co., Florida
Okeechobee Co., Florida
Bowden Fm., Jamaica
Esmeraldas Fm., Ecuador
Baja California, Mexico
Guaymas, Mexico
Sarasota Co., Florida
Collier Co., Florida
Collier Co., Florida
Caloosahatchee Fm., Florida
Caloosahatchee Fm., Florida
Waccamaw Fm., North Carolina
Bermont Fm., Palm Beach Co., Florida
Mississippi delta, Louisiana
Bowden Fm., Jamaica
Jamaica
Pinecrest Beds, Sarasota Co., Florida
Waccamaw Fm., South Carolina
Waccamaw Fm., North Carolina
Bahamas
(Olsson and Harbison, 1953)
Bowden Fm., Jamaica
San Felipe, Baja California, Mexico
San Felipe, Baja California, Mexico
Gulf of California, Baja California, Mexico
Panama
Gulf of California, Baja California, Mexico
Esmeraldas Fm., Ecuador
No. of
specimens
7
68
27
11
100
34
15
Max.
height
(mm)
39.83
41.46
38.07
47.34
32.41
36.86
34.48
38.37
26.91
31.58
26.87
36.24
38.76
22.07
27.49
26.03
35.35
12.97
37.43
47.99
52.17
57.15
52.31
55.84
130 ROOPNARINE
est Pliocene. Caloosahatchian descendants
of Early Pliocene conspecifics remain quite
large, all exceeding 30 mm in height. New
species, for example Puberella morsitans
(Olsson & Harbison), are also large. In the
Atlantic Gatunian, however, species from the
Upper Pliocene Bowden Formation do not
exceed 25 mm in height. The species in this
formation, Chionopsis woodwardi, Puberella
sawkinsi and Lirophora hendersoni (Dall),
range in size from 20-25 mm.
Species in the Caribbean today remain rel-
atively small. The two most common species,
Chione cancellata and Lirophora paphia (Lin-
naeus) rarely exceed 30 mm in height. Nota-
ble exceptions occur in upwelling areas (Ver-
meij, 1978), for example off the coast of
northern Venezuela. This region yields large
specimens of C. cancellata. The largest one
measured in this study was 35.35 mm in
height. Species from the coastal waters of
the United States, however, are comparable
in size to Early and Late Pliocene Caloosa-
hatchian species. All species examined had
specimens over 30 mm. Palmer (1927) de-
scribes a specimen of the Caribbean species
Puberella pubera with a valve height of 51
mm, but does not give detailed locality infor-
mation.
It is noteworthy that no Recent species in
neither the Atlantic Gatunian nor the
Caloosahatchian Provinces exceeds 40 mm
in height. Table 4 indicates that there were at
least four Early Pliocene species (Chione
erosa Dall, Chionopsis tegulum, Chionopsis
procancellata, and Panchione ulocyma), and
three Late Pliocene Caloosahatchian species
(Chionopsis procancellata, Puberella cribraria
(Conrad), and P. morsitans) which did exceed
40 mm in height. All these species are ex-
tinct, and the subgenera Chionopsis and
Panchione are today paciphilic. In contrast,
Recent Pacific chionine species measured
for this study are very large. Chione californ-
iensis Broderip and С. undatella both attain
heights in excess of 50 mm, while the spe-
cies Chionopsis amathusia and C. gnidia
(Broderip & Sowerby) have maximum sizes of
41.61 mm and 75.15 mm respectively. One
specimen of Puberella pulicaria (Broderip)
from the Gulf of California measured 35.50
mm. Moreover, the subgenera that have
evolved in, and are restricted to the Eastern
Pacific, Chionista, lliochione and Chione tu-
mens are also quite large; for example,
Chionista cortezi (Carpenter), 64.70 mm;
Chionista fluctifraga, 49.04 mm; lliochione
£ 80
> ст © N
© © о o
Maximum valve height (m
©
©
FIG. 18. Maximum valve heights of chionine spe-
cies during the late Neogene. Open circles—Atlan-
tic Gatunian region; filled circles—Caloosahatch-
ian Province; open triangles—Pacific Gatunian
region.
subrugosa, 34.26 mm; Chione tumens, 39.01
mm. In general, Pacific species are larger
than their Atlantic congeners. Figure 18 sum-
marizes these data, and Figure 19 presents
the data for individual subgenera.
DISCUSSION
Evolution of Chione
Assuming that the paleontological record
of chionine species is reasonably well docu-
mented, the common ancestor of the Chione
subgenera, as well as Anomalocardia, had
evolved by the Late Eocene, possibly earlier.
Some subgenera bear morphological resem-
blances to others, for example Lirophora and
Panchione, but all appear in the fossil record
essentially fully developed, with taxon-defin-
ing synapomorphies present.
Within the entire clade (comprising Anom-
alocardia, Chione, Chionista, lliochione, Liro-
phora, and Panchione) there are examples of
convergent and parallel evolution exhibited
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 131
Chione
50
45
40
A
44 pancuione
©
©
»
Chionopsis 0
36 Lirophora
Е Sr
28
24 Е
20
42
40
32 - Puberella
FIG. 19. Subgeneric breakdown of data presented in Fig. 18. Circles—Pacific Gatunian region; triangles—
Atlantic Gatunian region; squares—Caloosahatchian Province. Note extinction of Chionopsis and Pan-
chione in Atlantic regions.
132 ROOPNARINE
by related taxa inhabiting similar habitats.
Two characters, however, define the clade
strongly and consistently. The smooth con-
dition of the right middle cardinal tooth dis-
tinguishes the clade from the outgroups Mer-
cenaria, Protothaca and Timoclea, as well as
Chionopsis and Puberella. These outgroup
taxa have teeth that are grooved, with the
exception of Mercenaria, which has a bifid
tooth. That condition is convergent with the
bifid tooth of Chionista. The concentric
sculpture is also diagnostic of the clade. The
distal edge of the concentric lamellae tend
to be smooth in all members of the clade,
except Chione chipolana, which has a rein-
forcing ridge along the lamellar edge, and C.
cancellata, which has a sharp edge (the com-
mon condition in Chione s.s.). The common
outgroup condition (plesiomorphic) is sharp
edged lamellae. Puberella however, also has
distally reinforced lamellae.
The two subclades are likewise well de-
fined. The Chione subclade is defined prima-
rily by three characters; an extremely shallow
pallial sinus, vertical orientation of the con-
centric lamellae, and a narrow left anterior
cardinal tooth. Taxa of the Lirophora sub-
clade on the other hand, have four strong,
diagnostic synapomorphies; a short pallial si-
nus, the dominance of concentric sculpture
relative to radial sculpture, folding of the con-
centric sculpture, and the possession of a
rugose nymph. The characters that define the
two subclades contrast strongly between
them, but it is interesting to note that only two
characters are the common strong apomor-
phic definitions of these two clades. Consid-
ering the levels of homoplasy in characters
that distinguish the subclades, it would be
imprudent to make inferences of relationship
and clade membership based on analysis of
a single or few characters.
A note of caution must be expressed at this
point against the use of seemingly consis-
tent, functionally significant single characters
as descriptors of phylogeny or taxonomic
classification. The possession of a function-
ally significant character state by several taxa
could be indicative of common ancestry, but
might also signify the homoplastic nature of a
trait that is in “high demand.” Fisher (1981,
1985) argued this with respect to the multi-
ple, independent evolution of functionally
significant traits. An example relevant to this
study is the condition of the nymph. The
nymph as described above is a platform on
the valve margin, just posterior to the poste-
rior cardinal tooth. It is an attachment site for
the elastic ligament between both valves,
and is typically smooth. In several chionine
taxa (Мегсепапа and the entire Lirophora
subclade), however, the nymph is noticeable
rugose. The distinct difference between the
two states of this character has made it very
useful in defining species and subgeneric
boundaries. Moreover, its role as an attach-
ment site for the ligament has lent it some
notion of functional significance, though how
smoothness and rugosity may affect liga-
ment biomechanics is unknown. Harte
(1992a) used the roughened nymph of Liro-
phora kellettii (revised in this study to Pan-
chione kellettii) as the basis for assigning the
species to Mercenaria. Implicit in her study
was the untested assumption that the tradi-
tional taxonomic genus Chione is a mono-
phyletic clade, provided that the genus Mer-
cenaria is nested within it. The phylogenetic
analysis presented here does not support the
nesting of Mercenaria with Chione subgen-
era, and has demonstrated therefore that
Chione is not а monophyletic genus, and that
the roughened nymphs of Lirophora and
Mercenaria are most likely homoplastic.
Overall, the entire Chione clade has under-
gone tremendous diversification since the
Early Oligocene. There are 90 species re-
corded in this paper. Perhaps even more
striking is the number of distinct morpholo-
gies, or “bauplans” (as defined by Hall,
1992), that are nested within the clade.
Though the entire clade is supported by sev-
eral characters, for example prominent con-
centric sculpture, the subgenera as men-
tioned earlier can all be distinguished easily
from each other. These terminal taxa are not
necessarily distinguished by autapomorphic
characters, but more often by possessing
unique combinations of synapomorphic
character states. Moreover, the taxa, or the
synapomorphic combinations that they rep-
resent, appear in the fossil record abruptly
and fully defined. In general, the original sy-
napomorphic combinations still define the
subgenera in the Recent, and allow the easy
assignment of species to proper subgeneric
clades. It would seem therefore that above
the species level the rate of character evolu-
tion in the Chione clade was initially very
high, probably during the Late Eocene, but
character innovation has since stabilized or
fallen to near zero. To-date there is very little
fossil evidence to shed light on the origins of
individual subgenera.
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 133
Taxonomic Revision
Revision of a taxonomic classification on
the basis of phylogenetic analysis should
meet at least two important criteria. First, the
new classification must reflect the sister-
group relationships implied by the analysis
(deQueiroz & Gauthier, 1992). Secondly, re-
visions of the existing classification should
be minimized, with the only alterations of
rank and taxon membership being those ne-
cessitated by the phylogenetic analysis
(Wiley et al., 1991). This criterion ensures that
rank-based studies are affected as little as
possible, but are consistent with the phylo-
genetic analysis. Rank taxa would therefore
represent monophyletic clades, and as such
would remain convenient, phylogenetically
meaningful units for use in studies of diversi-
fication and extinction. The simplest method
for achieving these goals is to convert the
taxonomic classification to the tree that it
supports, and then evaluate the logical con-
sistency of the taxonomic tree with the phy-
logenetic tree. Changes made to the taxo-
nomic tree to make it logically consistent with
the phylogenetic tree are subsequently trans-
lated to modifications of the rank taxonomic
classification (Wiley et al., 1991).
The three cladograms and the resulting
consensus trees all show that the genus
Chione is paraphyletic because of the recog-
nition of Anomalocardia as a separate genus.
If the prevailing classification is converted to
a phylogenetic tree (Fig. 20), it is immediately
obvious that it is logically inconsistent with
the consensus tree. Not only does the taxo-
nomic classification not imply relationships
among the Chione subgenera, but there are
no hypotheses concerning the relationships
among the chionine genera. The simplest
resolution of the problem is the revision of
Chione. Harte (1992a) suggested that Merce-
naria be subsumed under Chione as a new
subgenus. That solution, however, would
disturb the long-standing understanding of
Mercenaria as a genus. For reasons of con-
sistency, it would also necessitate the inclu-
sion of Anomalocardia as a Chione subge-
nus. Such alterations would violate the
criterion of minimal changes outlined above.
Alternatively, several Chione subgenera
could be elevated to genus rank. Based on
the treatment of several of these subgenera
as genera by previous authors, the elevations
would minimize the changes necessary to
convert the taxonomic classification to a phy-
Anomalocardia
Mercenaria
Protothaca
Timoclea
Chione
Chionista
Chionopsis
Iliochione
Lirophora
Panchione
Puberella
Mercenaria
Puberella
Chionopsis
Protothaca
Timoclea
Chionista
Chione
Anomalocardia
Iliochione
Lirophora
Panchione
FIG. 20. Upper figure illustrates the maximum
amount of phylogenetic information that can be
derived from the traditional rank classification.
Lower figure is the consensus tree of this study,
showing genera only.
logenetic one. As discussed earlier, Wood-
ring (1982) and Ward (1992) both consid-
ered Lirophora to be of genus rank, and
Olsson (1964) treated Chionopsis similarly.
Keen (1969) also elevated Panchione to ge-
nus rank. Elevations of these subgenera to
genus rank would therefore not be uncon-
ventional.
The changes required to convert the taxo-
nomic classification to a phylogenetic classi-
fication begin with the exclusion of Timoclea
from consideration as a subgenus of Chione
and recognition of it as a distinct genus.
Next, the subgenera Chione, Lirophora, Шо-
chione, and Panchione are elevated to genus
rank. Puberella, originally designated a sub-
genus of Chionopsis (Fischer-Piette & Vuka-
dinovic, 1977), is elevated to generic rank. In
the monophyletic clade comprising Liro-
phora, Anomalocardia, lliochione and Pan-
chione, both lliochione and Panchione must
be considered separate genera, due primarily
134 ROOPNARINE
TABLE 5. Comparison of traditional classification (left columns) to new phylogenetically
based classification. Subgenera listed on right are genera sensu stricto.
OLD CLASSIFICATION
NEW CLASSIFICATION
GENUS SUBGENUS
Chione Chione
Chionista
Chionopsis
lliochione
Lirophora
Panchione
Puberella
Anomalocardia
Mercenaria
Protothaca
Timoclea
to the uncertain position of /liochione. The
alternative would require the subsuming of
both genera, plus either Lirophora or Anom-
alocardia, into a large genus classified as ei-
ther Lirophora or Anomalocardia, on the ba-
sis of historical precedence. The subgeneric
rank of Chionista is supported by the reclas-
sification. Table 5 compares the old taxo-
nomic classification with the new phyloge-
netic one. lt is important to note here the
number of taxa (genera) which were formerly
subsumed in the paraphyletic Chione, and
the exclusion of other genera on the basis of
autapomorphies, not patterns of relationship.
Pliocene Extinctions
Since Woodring's (1966) initial claim, the
general picture of late Neogene molluscan di-
versity in tropical America has been one of
high Pliocene extinction in the tropical West-
ern Atlantic compared with the Eastern Pa-
cific (Vermeij, 1978; Vermeij 4 Petuch, 1986).
Coupled with this is the documentation of ex-
tensive contemporaneous extinctions in the
subtropical waters off the southeast United
States (Stanley, 1986). Several mechanisms
have been suggested to explain the extinc-
tions, the most commonly cited ones being
disruption of planktonic productivity levels
(Vermeij, 1978; Vermeij & Petuch, 1986; All-
mon et al., 1993) and Northern Hemisphere
cooling (Stanley, 1986). It has also been ar-
gued that the extinctions were in fact faunal
turnovers, and that losses of diversity have
been overcompensated for by new origina-
tions and invasion (Allmon et al., 1993; Jack-
son et al., 1993; see also Vermeij & Rosen-
GENUS SUBGENUS
Chione Chione
Chionista
lliochione
Lirophora
Panchione
Puberella
Chionopsis
Anomalocardia
Mercenaria
Protothaca
Timoclea
berg, 1993). Jackson et al.'s (1993) data
(comprising subgeneric rank taxa) also sup-
port the initiation of the extinctions at 2.4 Ma,
well into the Late Pliocene.
The history of the chionine taxa considered
in this paper agree in general with the sce-
nario constructed by Jackson et al. (1993),
with several exceptions. During the entire
Pliocene, extinction of Atlantic chionine spe-
cies (restricted from here on to species be-
longing only to the former Chione subgenera
and Anomalocardia) exceeded origination
(Fig. 12). Origination decreases in the Pacific
during the Pliocene, but is never outweighed
by extinction. This observation, and a high
origination rate in the Pacific during the Pleis-
tocene and Holocene, result in a higher total
diversity of chionine species in the Eastern
Pacific today compared to the Western At-
lantic. Two new supraspecific taxa, Chionista
and lliochione, also first appear in the Eastern
Pacific during the Pliocene. Overall, the ex-
tinction level of chionine species during the
entire Pliocene was 82.6% in the tropical
Western Atlantic, but only 38.5% in the Pa-
cific Gatunian (Fig. 13). Today there are 21
chionine species in the Eastern Pacific, ver-
sus 13 in the Western Atlantic (Chione can-
cellata is considered to comprise two distinct
“morphological” species, based on Roop-
narine, 1995). Of the five subgenera present
in both regions during the Pliocene, all sur-
vive in the Eastern Pacific today, but only
three in the Western Atlantic. The conclu-
sions to be drawn from this study therefore,
are that extinction of chionine species during
the Pliocene was significantly higher in the
Western Atlantic than in the Eastern Pacific,
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 135
and the loss of diversity was not compen-
sated for by new originations.
The timing of a large portion of the extinc-
tion remains ambiguous. Of Early-middle
Pliocene Chione species in the Western At-
lantic 45% were absent from the Late
Pliocene (Fig. 15). Extinction in the Eastern
Pacific at that time was a slightly higher 57%
(though species diversity was lower than in
the Western Atlantic) During the Late
Pliocene, however, extinction in the Western
Atlantic increased to 66.7%, but fell in the
Eastern Pacific to 11.1%. Dating the extinc-
tion to the Late Pliocene on the basis of these
summary calculations would be incorrect,
though, because the pattern is not reflected
by all the individual subgenera. Lirophora and
Panchione exhibit much higher extinction in
the Early-middle Pliocene (60% and 100%
respectively) than do the other subgenera
(Fig. 16). It therefore seems that there were at
least two episodes of extinction.
The first extinction episode, in the Early-
middle Pliocene, was associated primarily
with the subgenera Lirophora and Pan-
chione. The extinctions were also concen-
trated in the Atlantic Gatunian region. The
reason(s) for the differential extinction among
the subgenera is not obvious. Lirophora and
Panchione are nested within a subclade sep-
arate from the subclade to which Chione be-
longs, but the characters distinguishing the
two subclades currently have limited func-
tional interpretations. There are furthermore
no obvious ecological differences among the
subgenera. All inhabit fairly shallow, coarse
to medium-grained sediments, and are sym-
patric today. Lirophora and Panchione prob-
ably dwell deeper in the sediment than does
Chione, as evidenced by their deeper pallial
sinuses. Anomalocardia, however, disrupts
any phylogenetic pattern because it experi-
ences minimal levels of extinction. The
greater proportion of Anomalocardia diversity
resided in the Caloosahatchian Province, not
the Atlantic Gatunian region, unlike Lirophora
and Panchione, and is suggestive of and
consistent with a geographic extinction pat-
tern. This possibility is explored more fully
below.
Mechanisms of Extinction
Two expectations of extinction caused
by cooling would be more severe extinction
of Atlantic Gatunian species relative to
Caloosahatchian species, and the higher sur-
vival of eurythermal (biprovincial) species
(Stanley, 1984, 1986). According to Cronin's
(1991, 1993) data, the Northern Hemisphere
cooling event reported by Stanley (1986) ac-
tually began 2.6-2.4 Ma. This event coin-
cided with, and was probably causative of, a
major regression recorded on the Atlantic
Coastal Plain, and allows the categorization
of the Pliocene deposits into Early-middle
and Late Pliocene groups. Of the species in
the Atlantic Gatunian during the Early-middle
Pliocene 40% did not survive into the Late
Pliocene (Fig. 17). The extinction level in the
Caloosahatchian Province at that time was
only 18.75%. Stanley's prediction would
therefore seem to be supported. Of the Late
Pliocene species in the Atlantic Gatunian and
Caloosahatchian 36.4% and 50% respec-
tively were extinct by the Early Pleistocene.
At this level of the analysis, the data cannot
reject Stanley's cooling hypothesis. How-
ever, the possibility that the surviving species
were eurythermal cannot be entertained, be-
cause there were no biprovincial chionine
species in the Late Pliocene. A possible ex-
ception would be Chione cancellata, except
that, as noted above, the Caloosahatchian
and Caribbean forms may be separate spe-
cies. The Caloosahatchian form first appears
in the Upper Pliocene/Pleistocene Wacca-
maw Formation of South Carolina, and has
not been documented further south than the
Florida peninsula. Therefore, no evidence ex-
ists to support the higher survival of euryther-
mal species, nor the survival of Caloosahat-
chian species by southward migration.
The complete pattern of Pliocene extinc-
tion of chionine species does not support a
hypothesis of cooling as a mechanism. Ex-
tinction is higher in more tropical areas dur-
ing or soon after the initiation of cooling, but
the higher level is due to the non-random ex-
tinction of Lirophora and Panchione species.
By the Late Pliocene, extinction was distrib-
uted fairly evenly among all the subgenera. It
is interesting to note, however, that the two
Recent paciphilic chionine genera, Chionop-
sis s.s. and Panchione, were distributed
throughout the Atlantic Gatunian, but only as
far north as Florida during the Pliocene.
These are the only two taxa to suffer com-
plete extinction in the Atlantic. Their extinc-
tions do not appear to support a hypothesis
of cooling though, because these taxa failed
to find refuge by migrating equatorward.
The indirect test of declining planktonic
productivity as a mechanism of extinction
136 ROOPNARINE
yields more intriguing results. All Early-mid-
dle Pliocene Atlantic Caloosahatchian spe-
cies exceeded 35 mm in maximum valve
height. Only two of eight contemporary At-
lantic Gatunian species, Chionopsis tegulum
and Panchione mactropsis, exceeded 35
mm, and these in fact exceeded 40 mm. Only
one of the Caloosahatchian species did not
survive beyond the middle Pliocene. Three of
the Atlantic Gatunian species survived into
the Late Pliocene, but they were all small,
being less than 25 mm in height. Caloosahat-
chian species, survivors and new Late
Pliocene species on the other hand remained
relatively large, all exceeding 30 mm in max-
imum valve height, and three of six exceed-
ing 40 mm. Recent Caloosahatchian species
regularly exceed 30 mm in valve height, but
none are known to attain heights of 40 mm or
more. Caribbean species have remained
small, generally not exceeding 25 mm. Nota-
ble exceptions occur in areas of upwelling,
and hence relatively high planktonic produc-
tivity. Recent Pacific species commonly at-
tain heights in excess of 50 mm (Fig. 19);
Chionopsis gnidia is the largest described
chionine species dealt with in this paper. In
summary, size distributions have not
changed very much in the Caloosahatchian
Province since the Early Pliocene, despite
changes in species composition. In the Ga-
tunian Province though, the change has been
more dramatic. Large species in the Atlantic
Gatunian region did not survive into the Late
Pliocene. Species size distribution there re-
mains small because of the differential ex-
tinction of the larger Early-middle Pliocene
species, and apparently because no large
(> 35 mm) species have evolved in that re-
gion since. The opposite is true of the Pacific
Gatunian, or Panamic Province. Post Early-
middle Pliocene species there tend to be very
large, perhaps a consequence of the wide-
spread coastal upwelling in that region.
Given the equivocal nature of the results of
the above tests, any hypothesized cause(s)
of chionine extinction during the Pliocene in
the Western Atlantic is speculative. More-
over, the data are limited to only those gen-
era examined, and the availability of material
documented in the field, museum collec-
tions, and the literature. Regardless, the pat-
tern of extinction can be explained tentatively
by current geological data which appear to
support a decline in productivity, at least in
the Early-middle Pliocene.
The loss of Early-middle Pliocene large-
and small-bodied species from the Atlantic
Gatunian, representing 40% of the species,
contrasts strongly with the contemporary
Caloosahatchian. Only 18.75% of Early-mid-
dle Pliocene Caloosahatchian species did
not survive into the Late Pliocene, and there
is no reduction in overall body size. There is
therefore an indication of an Early-middle
Pliocene episode of extinction in the Atlantic
Gatunian that did not have a great impact on
the Caloosahatchian Province. The Late
Pliocene extinctions in the Atlantic Gatunian
are matched in severity, however, by the Late
Pliocene Caloosahatchian extinctions, but
there is no change in overall body size in ei-
ther province. lt is therefore possible that
there were two episodes of extinction in the
tropical Western Atlantic during the Pliocene
(Petuch, 1995).
The earlier extinction in the Atlantic Gatu-
nian probably followed the final closure of the
Panama seaway (~3.5 Ma). Final closure oc-
curred during the earliest Pliocene when the
shallowing of the seaway was already dra-
matic (< 100 m depth, Duque-Caro, 1990;
see also Coates et al., 1992). The shallowing
undoubtedly resulted in the fragmentation of
once contiguous and widespread popula-
tions, but perhaps more importantly, it also
changed the oceanographic configuration of
the Caribbean region. It has not yet been de-
termined how changes in circulation and the
decline and eventual termination of flow from
the Atlantic to the Pacific may have affected
local diversity. The presence of seasonal cool
water in the Caribbean during the middle
Pliocene (Cronin, 1991) unlike today, the ex-
istence of relict communities in the Carib-
bean in areas of upwelling today (Petuch,
1982), and the loss of large bodied species
from the Atlantic Gatunian (this paper) how-
ever, all suggest a decline in planktonic pro-
ductivity. Interestingly, though, Lower-middle
Pliocene deposits in the Caloosahatchian
Province show no signs of significant extinc-
tion or loss of large bodied species. This
would suggest that the extinctions did not
extend to, or did not affect the Caloosahat-
chian Province significantly.
The Late Pliocene extinctions of the Atlan-
tic Gatunian and Caloosahatchian provinces
coincide with the initiation of Northern Hemi-
sphere cooling (2.5-2.4 Ma) (Stanley, 1986;
Cronin, 1991, 1993). Neither extinction, how-
ever, exhibits pattern expected of cooling
scenarios. The only noticeable difference be-
tween Late Pliocene and Recent faunas, be-
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 137
sides the lower diversity of modern faunas, is
the absence of very large-bodied species
(> 40 mm) in the Recent. Large species, or
large specimens (30-35 mm), survive in the
Caloosahatchian region, and in areas of up-
welling in the Caribbean. This observation
suggests that declining planktonic productiv-
ity may again have been a factor, but perhaps
not the only, or primary one.
SUMMARY
(1) Phylogenetic analysis shows that the
genus Chione as currently defined is para-
phyletic. À monophyletic clade comprises
the genera Chione (excluding the subgenera
Chionopsis and Puberella), and Anomalocar-
dia. Chione has been revised in order to con-
struct a taxonomic classification that is con-
sistent with the results of the phylogenetic
analysis. The genus was revised and re-
placed by the genera (formerly subgenera)
Chione (s.s.), Chionopsis, Lirophora, Шо-
chione, Panchione and Chionista. The treat-
ments of Chionopsis as a genus (Olsson,
1964), similarly Lirophora (Woodring, 1982;
Ward, 1992), and the consideration of Pan-
chione as a subgenus separate from Liro-
phora (Keen, 1969), support the revision.
(2) Sculptural characters, primarily the re-
tention of radial sculpture, and the marginal
elaborations of concentric sculpture, are di-
agnostic of the ‘‘Chione subclade” (compris-
ing the genera Chione and Chionista). The
“Lirophora subclade” is defined more
strongly by a combination of characters de-
scribing internal as well as external valve
morphology, most notably aspects of con-
centric sculpture and nymph rugosity.
(3) During the late Neogene the origination
of new chionine species rose steadily in the
Eastern Pacific. The number of last appear-
ances increases briefly during the Pliocene,
but never exceeds the number of first ap-
pearances. Of Early-middle Pliocene species
in the Atlantic Gatunian Province 40% do not
survive into the Late Pliocene, compared with
18.75% of Early-middle Pliocene Caloosa-
hatchian species. The higher extinction in the
Atlantic Gatunian region is accounted for pri-
marily by the seemingly non-random extinc-
tion of species assigned to the genera Liro-
phora and Panchione. During the Late
Pliocene however, 36.4% and 50% of spe-
cies in both the Atlantic Gatunian and
Caloosahatchian Provinces respectively are
last appearances, and these extinctions
seem to be distributed randomly among the
surviving genera. This wave of extinction be-
gan at least 2.5 million years ago.
(5) A hypothesis of cooling as a mechanism
of extinction is not supported by these data.
Extinctions caused by cooling would have
been restricted to the Late Pliocene because
of the late date of the onset of Northern
Hemisphere refrigeration (Cronin, 1991,
1993). Extinction is not more severe in the
fully tropical Atlantic Gatunian Province than
the sub-tropical Caloosahatchian. Moreover,
there is no evidence that Caloosahatchian
species survived the crisis by southward mi-
gration. Some equivocal support might be
provided by the observation that neither of
the two paciphilic genera examined, Chion-
opsis (s.s.) and Panchione, extended further
north than Florida during the Pliocene.
(6) The loss of all large bodied (> 35 mm)
species from the Early-middle Pliocene At-
lantic Gatunian Province is consistent with a
hypothesis of declining planktonic productiv-
ity. During the Late Pliocene, however, body
size did not seem to be a very discriminating
factor, since small bodied Atlantic Gatunian
species also suffered extinction. Modern
species have maximum valve heights, in the
Caloosahatchian and Caribbean, ranging
from 25 to 35 mm. On the other hand, Late
Pliocene Caloosahatchian species exceed-
ing 40 mm in maximum valve height suffered
100% extinction. Therefore, it seems that
large bodied species did suffer differentially
higher levels of extinction during the
Pliocene. The mechanism responsible is hy-
pothesized to be a disruption of planktonic
productivity patterns and levels. Additional,
indirect evidence is provided by the Late and
post-Pliocene evolution of very large chion-
ine species in the upwelling-rich Panamic
Province.
ACKNOWLEDGMENTS
| wish to thank the following individuals for
guidance throughout the completion of this
project: Geerat Vermeij, Sandra Carlson and
Mark Patterson. The following individuals
were also helpful in providing comments and
assistance: Jay Schneider, Kim Driver, Scott
Gardner, Stanley Bursten, William Coles,
Eleanor Villar and Richard Cowen. | thank R.
Bieler (Field Museum of Natural History), H.
Lescinsky (University of California, Davis), R.
138 ROOPNARINE
Portell (Florida Museum of Natural History),
G. Vermeij (University of California, Davis)
and E. Vokes (Tulane University) for the kind
loan and contribution of specimens. This
work was supported by grants from the De-
partment of Geology, University of California
Davis, The Center for Population Biology,
University of California Davis, the Lerner-
Gray Fund for Research, Sigma Xi and the
Paleontological Society.
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APPENDIX |
Left Valve
1. Depth of pallial sinus: O = greatly reduced
to absent; 1 = present but short; 2 = pallial
sinus deep, anterior end to beneath posterior
cardinal tooth; 3 = very deep, past beneath
posterior cardinal.
The most common condition of the pallial
sinus in chionine bivalves is moderately to
very deep. Examples can be found in the
genera Mercenaria and Timoclea. Many other
taxa, for example Lirophora, have very short
sinuses. An intermediate condition can be
found in Puberella and Chionopsis. Neither
Chione nor Petenopsis have a recognizable
pallial sinus.
2. Types of sculpture present: 0 = concentric
elements only; 1 = radial and concentric
elements, co-dominant; 2 = radials and con-
centrics, concentrics dominant, radials sub-
obsolete; 3 = radials and concentrics, both
sub-obsolete; 4 = radials and concentrics,
radials dominant.
There are two types of sculptural elements
in the Chioninae, radial and concentric. Both
forms are present and co-dominant in Chione
and Chionopsis (including Puberella). The
concentric elements in these genera are thin,
raised lamellae, whereas the radials are
raised and cordlike. The radials are absent in
Mercenaria, but this appears to be a derived
autapomorphic condition, not homologous
with the “concentric-dominant” sculpture of
Lirophora, Anomalocardia, lliochione and
Panchione; Mercenaria’s concentrics are of
the Chione and Chionopsis type. Lirophora,
Anomalocardia, /liochione and Panchione all
have greatly reduced or absent radial ele-
ments, and the development of thick, folded
concentric lamellae. Leukoma and Timoclea,
on the other hand, lack concentric elements
almost entirely, but have radial sculpture very
similar to the Chione and Chionopsis. The
sculptures of Chionista and Petenopsis seem
to be autapomorphic.
3. Spacing of concentric elements: 0 = close
(narrow spacing); 1 = widely spaced; 2 = wide
spacing on older, juvenile shell, but tending
to become more narrowly spaced with in-
creasing age.
4. Posterior development of concentric ele-
ments: O = flared in ventral direction; 1 =
weakly flared, but flattened; 2 = no flare.
The posterior end of concentric elements is
highly developed in many chionine taxa, pos-
sibly for functional reasons associated with
shallow burrowing.
5. Anterior development of concentric ele-
ments: 0 = no anterior projection; 1 = devel-
oped anteriorly; 2 = weak anterior develop-
ment.
The concentric elements are developed
anteriorly in many chionine taxa, and these
may aid during burrowing, probably acting as
ratchets, but this has not been rigorously
tested.
6. Structure of concentric elements: O = con-
centric elements smooth; 1 = ventral surface
of concentric elements bearing closely
packed vertical ribs; 2 = ventral surface of
concentric elements bearing widely spaced
vertical ribs; 3 = concentric elements present
only as raised structures on radial elements.
7. Distal edge of concentric elements: O =
sharp; 1 = smooth; 2 = smooth and rein-
forced.
The distal edges of the concentric ele-
ments are either thin and sharp, or thickened
NEOGENE EXTINCTION OF TROPICAL AMERICAN BIVALVES 141
and smooth. Some, as in Puberella, are thin,
but have a thickened, reinforced summit.
8. Orientation of concentric elements: O =
vertical; 1 = folded; 2 = step-like and
rounded.
This character describes the orientation of
concentric elements relative to the surface of
the valve.
9. Definition of escutcheon: 0 = escutcheon
developed and set off from rest of valve by
obvious ridge (''keel”); 1 = escutcheon de-
veloped but weakly keeled; 2 = escutcheon
not developed.
10. Width of anterior cardinal tooth: 0 = wide;
1 = narrow.
The anterior cardinal is either well devel-
oped and wide, as in Chione, or thin and
blade-like, as in Chionopsis.
11. Nymph: 0 = rugose; 1 = smooth.
The nymph, a platform posterior to the
posterior cardinal tooth, houses the ligament
in venerid species. The nymph is generally
smooth, but can have a roughened or “rug-
ose” surface. The state of this character is
sometimes given predominant weight in de-
termining chionine relationships (for exam-
ple, Harte 1992a), but nymph rugosity, of
variable morphology, is present in non-chio-
nine taxa, for example Pitar (Lamelliconcha)
Dall, 1902. Nymph rugosity is therefore ho-
moplastic at some levels.
12. Ventral margin crenulation: O = large; 1 =
fine; 2 = intermediate between 0 and 1, and
regular.
13. Lunule sculpture: 0 = numerous concen-
tric elements; 1 = few concentric elements; 2
= smooth; 3 = concentric elements with sub-
dominant radial ribs; 4 = sub-obsolete con-
centric and radial elements; 5 = numerous
radial ribs; 6 = lunule not developed.
14. Middle cardinal tooth morphology: O =
bifid; 1 = smooth; 2 = smooth, except for
dorso-ventral groove.
15. Posterior cardinal tooth shape: 0 =
straight; 1 = curved; 2 = weakly curved.
16. Shape of hinge plate margin: O = plate
very bowed beneath anterior cardinal tooth; 1
= plate weakly bowed, not obvious; 2 = plate
bowed, not exaggerated as in 0; 3 = plate
straight.
Many chionine species have hinge plates
that are noticeably bowed, or curved, be-
neath the anterior cardinal tooth. An extreme
example of this can be found in Mercenaria.
Other taxa have straight margins, and there is
a continuum between the two states.
Right Valve
17. Definition of escutcheon: 0 = escutcheon
developed, but not demarcated noticeably
from rest of valve surface; 1 = escutcheon
developed and demarcated from rest of valve
by keel; 2 = escutcheon not developed.
18. Sculpture of escutcheon: 0 = concentri-
cally sculptured; 1 = escutcheon smooth; 2 =
sub-obsolete concentric sculpture; 3 = es-
cutcheon absent;
19. Width of middle tooth: O = tooth narrow;
1 = tooth wide.
20. Condition of middle cardinal tooth: O =
tooth bifid; 1 = tooth grooved on dorsoventral
axis; 2 = tooth smooth.
21. Condition of posterior cardinal tooth: O =
tooth bifid; 1 = tooth grooved on dorsoventral
axis; 2 = tooth smooth.
22. Orientation of groove on posterior margin:
0 = groove just overlaps ventral tip of posterior
cardinal tooth; 1 = groove distal to posterior
cardinal tooth; 2 = groove abuts posterior car-
dinal tooth; 3 = groove overlaps posterior car-
dinal tooth significantly; 4 = groove absent.
This groove is present in many chionine
taxa on the right valve, and houses the pos-
terior margin of the left valve when the shell is
closed.
23. Condition of radial ribs between concen-
tric sculpture: O = no radial ribs present; 1 =
radial ribs prominent; 2 = radial ribs present
but fine, tending to become obsolete.
General Shell Morphology
24. Escutcheon symmetry: 0 = escutcheon
symmetric between valves; 1 = escutcheon
asymmetric between valves; 2 = escutcheon
absent.
The escutcheon is generally of different
142 ROOPNARINE
morphology between valves in chionine taxa,
but is sometimes identical.
25. Development of valve surface near pos-
terior margin: O = identical to rest of valve
surface; 1 = sculpture and valve surface dif-
ferentially developed as posterior margin ap-
proached.
Some taxa have significant changes of
sculpture towards the posterior margin.
Much of the change is in concentric sculp-
tural morphology, as in Panchione mactrop-
sis, where the folded concentric sculpture
becomes lamellar. //iochione has an indenta-
tion of the valve.
APPENDIX II
(see APPENDIX | for explanation of numerical codes)
“9”= missing data
Chione cancellata
Chione chipolana
Chionopsis amathusia
Puberella cribraria
Lirophora athleta
Lirophora victoria
Panchione mactropsis
Panchione ulocyma
lliochione subrugosa
Chionista fluctifraga
Petenopsis tumens
Mercenaria mercenaria
Anomalocardia flexuosa
Protothaca asperrima
Timoclea marica
Anomalocardia auberiana
0120210000103221001212110
1100212000106121001212110
2110110011101011110101101
2100112001100020000110210
1002001101012121010213001
1000001101912121019919001
1201211101012221011210201
1202211101012223011210201
1202011101012101011223201
2302021120125002231004121
0112011200112121011222100
2000000000000000000000010
0202001111012103020221001
2402030001114201011112111
3401030001104103121124100
1202021111011101021221201
APPENDIX III
Synapomorphies for interior nodes, cladogram #1. Results of both ACCTRAN and DELTRAN
routines are listed. Interpret listings as (x:y) = (character:state). ACCTRAN
Node ACCTRAN
117 052 720) 13:3, 18:0, 241
18 1:0, 8:0, 15:2,.25:0
19 10:0
20 2:0, 6:0, 19:0, 23:0
21 5:2, 14:2, 22:0
22 15:2
23 6:0, 9:1, 18:25 22:1
24 21:2
25 Wed РО, ra 22:3 23:2
26 1:1, 8:1, 20:2
27 2:4, 6:3, 13:4
28 4224182 19:1, 19:1, 21 22:2
Synapomorphies
DELTRAN
4:0, 5:2, 18:0, 24:1
OMIS 292520)
10:0
2:0, 6:0, 23:0
5:2, 14:2, 22:0
15:2
9:1, 18:2, 22:1
24:2
1:1, 2:2, 8:1, WO} 12a 2223232
TA 82 20:2
2:4, 6:3, 13:4
4:2, NAA AG Al 21:1, 22:2
29 13:1, 16:1, 18:1,.22:1,.23:1,.24:0, 25:1 16:1, 18:1, 23:1, 24:0, 255]
MALACOLOGIA, 1996, 38(1-2): 143-151
THE GENITAL SYSTEM OF ACOCHLIDIUM FIJIENSE (OPISTHOBRANCHIA:
ACOCHLIDIOIDEA) AND ITS INFERRED FUNCTION
Martin Haase' & Erhard Wawra?
ABSTRACT
The genital system of the adolescent-gonochoric freshwater opisthobranch Acochlidium
fijiense is described from histological serial sections of five individuals and dissection of a sixth
animal in full detail. The penis has a characteristic armature consisting of an ascending spiral
of chitinous spines on the edge of the glans. The basal finger in association with the parapros-
tate probably functions as stimulatory organ analogous to the gypsobelum of pulmonate gas-
tropods. The presence of sperm in the haemocoel and the kidney of one specimen and the
penial armature suggest that A. fijiense transfers sperm through hypodermic impregnation. The
most peculiar feature is the connection of the genital system with the digestive system. A duct
with unknown function connects the digestive gland with the distal gonoduct. In addition, in one
individual the ampulla which stores autosperm had an opening into the digestive gland. This
opening is interpreted as a temporary structure established only when required in order to
digest excess autosperm, thus compensating the lack of a gametolytic gland. However, it
cannot be ruled out that this connection seen in a single individual was an abnormality.
Key words: Acochlidium, genital system, hypodermic impregnation, Opisthobranchia, sperm
transfer, stimulatory organs
INTRODUCTION
The vast majority of opisthobranch gastro-
pods are marine. Up to now only seven spe-
cies are known from freshwater habitats.
These seven species all belong to the order
Acochlidioidea. All marine acochlidioidean
species are smaller than 5 mm. All but one
species of the freshwater forms, on the other
hand, exceed 15 mm. The exception is the
Caribbean Tantulum elegans Rankin, 1979,
which lives interstitially (Rankin, 1979). The
large species—Strubellia paradoxa (Strubell,
1892) and five species of the genus Acoch-
lidium Strubell, 1892 [Following Wawra (1989),
we use aconservative classification and reject
Rankin’s (1979) taxonomic splitting.|—occur
on islands in the Pacific region (Haynes &
Kenchington, 1991). Despite the size of the
Pacific freshwater species, which would make
anatomical investigation and maintenance
and observation in aquaria rather easy com-
pared to small, interstitial snails, relatively little
is known on both their anatomy and biology.
The present study gives a detailed anatomical
description of the genital system of A. fijiense
Haynes & Kenchington, 1991, and allows in-
ferences on its function and the reproductive
biology of this species.
MATERIALS AND METHODS
Five individuals from the series of para-
types of A. fijiense from Vanua Levu, Fiji, de-
posited in the mollusc collection of the Mu-
seum of Natural History in Vienna by Haynes
& Kenchington (1991) under the inventory
number 84901 were embedded in Paraplast
and serially sectioned, one specimen at 7 um
and the remaining four at 10 um. The series
were stained with Heidenhain’s Azan. The
fixed (Bouin) snails measured 6.1 mm, 7.4
mm, 7.9 mm, and 8.03 mm respectively. One
specimen could not be measured because it
had the visceral hump turned down. These
individuals belonged to the largest among
the series of paratypes. In the following, they
will be referred to as snails number 1 to 5
beginning with the smallest animal. We do
not proceed on the assumption that these
snails represent a developmental sequence,
because they might have had contracted to
differing degrees at fixation. The genital sys-
tem was reconstructed using the computer
program PC3D of Jandel Scientific. The pe-
nis of a sixth paratype from the same lot was
dissected, critical point dried and investi-
gated by scanning electron microscopy
(SEM).
‘institut für Zoologie der Universität Wien, Althanstrasse 14, A-1090 Wien, Austria.
“Erhard Wawra, 3. Zoologische Abteilung, Naturhistorisches Museum Wien, Burgring 7, A-1014 Wien, Austria.
144 HAASE & WAWRA
FIG. 1. Reconstruction of the genital system ex-
cept gonad from dorsal. am = ampulla; de = ductus
ejaculatorius; dgd = distal gonoduct; fgm = female
gland mass; pd = paraprostatic duct; pg = prae-
ampullary gonoducts; ppr = paraprostate; pr =
prostate; ps = penial sheath; to = [presumptive (see
Discussion)] temporary opening of the ampulla into
the digestive gland; vd = vas deferens. Scale bar =
500 um.
FIG. 2. Distal genital system from latero-dorsal. dd
= duct connecting digestive gland and distal gono-
duct; de = ductus ejaculatorius; dgd = distal gono-
duct; fgm = female gland mass; pr = prostate; ps =
penial sheath; vd = vas deferens. Scale bar = 500
um.
RESULTS
The description of the genital system fol-
lows the route the gametes take from their
place of origin in the gonad to the genital
openings, that is from posterior to the ante-
rior end of the snail. The gonad is covered by
the lobes of the digestive gland and consists
of a large number of acini (Fig. 16). Oocytes
FIG. 3. Schematic representation of the genital
system and its connection with the digestive gland.
Accessory organs not drawn. am = ampulla; dd =
duct connecting digestive gland and distal gono-
duct; de = ductus ejaculatorius; dg = digestive
gland; dgd = distal gonoduct; fgm = female gland
mass; go = gonad; in = intestine; oe = oesophagus;
pg = praeampullary gonoduct; po = postampullary
gonoduct; pr = prostate; st = stylet; to = [presump-
tive (see Discussion)] temporary opening of the
ampulla into the digestive gland; vd = vas deferens.
FIGS. 4, 5. Origin of duct connecting digestive
gland and distal gonoduct. Increment between
Figs. 4 and 5 = 30 um. ao = aorta; dd = duct con-
necting digestive gland and distal gonoduct; dg =
digestive gland; dgd = distal gonoduct; in = intes-
tine; v = ventricle. Scale bars = 100 um.
and spermatocytes mature in the same aci-
nus. The gametes produced in these acini are
collected through a branching net of ciliated
preampullary gonoducts, which open into the
ampulla (Figs. 1, 16, 17). This ampulla con-
sists of a number of communicating cham-
bers. Its epithelium lacks cilia. The glandular
postampullary gonoduct connects the am-
pulla with the large female gland mass. The
duct enters the gland mass ventrally on the
left side. This gland mass has two histologi-
cally distinct portions, which probably func-
tion as albumen and mucous gland, respec-
tively. At the right side, the ciliated distal
gonoduct leaves the gland mass and
traverses the body wall to the anterior end,
where it opens close to the mouth (Figs. 1, 2).
There are two ducts branching off the dis-
tal gonoduct. Proximally, a short duct con-
nects the gonoduct with the digestive gland.
ACOCHLIDIUM FIJIENSE 145
FIGS. 6-11. Penis. 6. Apical view. Scale bar = 100 um; 7, 8. Lateral views. Scale bars = 100 um; 9. Large
Spine in epidermal sheath. Arrow indicates distal end of the sheath. Scale bar = 10 um; 10. Bulge of the
edge of the penial glans with small spines. Arrow indicates distal end of an epidermal sheath. Scale bar =
20 um; 11. Cross-section through the tip of the ejaculatory duct. Scale bar = 50 um. bf = basal finger; de
= ductus ejaculatorius; ep = epithelium; ps = penial sheath; st = stylet; th = thorn.
The junction of this short duct with the diges-
tive gland is close to the origin of the intestine
(Figs. 2-5). Distally, the vas deferens, ven-
trally attached to the penial sheath, leads
backwards to the prostate (Figs. 1-3, 14).
The muscular ductus ejaculatorius origi-
nates in the middle of the ventral side of the
prostate (Fig. 2). It enters the muscular penis
at its base after several coils between the
lobes of the prostate and the paraprostate
(see below) and around the penis (Figs. 1, 8,
14, 15). Distally, the ductus ejaculatorius
leaves the penis at the left side and rests on
its external wall (Figs. 6, 8, 12, 13) (In snail no.
3 the ductus ejaculatorius was completely re-
tracted into the penis.). The opening through
146 HAASE & WAWRA
de —
Cr .r
x ae
See eo eS ee =
E
=
\
FIG. 12. Schematic representation of the penis.
Chitinous elements are solid black. bf = basal fin-
ger; de = ductus ejaculatorius; ip = intrapenial
gland; pd = paraprostatic duct; ppr = paraprostate;
pr = prostate; th = thorn.
which the ductus ejaculatorius exits the penis
is guarded by a chitinous thorn (Figs. 7, 8, 12,
13). This thorn is associated with a gland Iy-
ing in the penis, which is hereafter referred to
as intrapenial gland (Fig. 12). At its tip, the
ductus ejaculatorius bears a chitinous stylet
which is in fact a groove closed by epithelium
(Fig. 11). Basally, at the dorsal side, the penis
bears a finger, the basal finger, armed with a
corneous stylet (Figs. 6, 7, 12). This hollow
stylet is connected with a gland, which we
call paraprostate because of its position ven-
tral of the actual prostate, by the parapros-
tatic duct (Figs. 1, 14, 15). The glans penis is
armed with chitinous spines, too (Figs. 6-10,
12). Two types of spines in an ascending spi-
ral on the edge of the glans can be distin-
guished. This spiral comprises almost an en-
tire whorl. In the specimen dissected, 12
large spines formed the lower semi-circle
and 24 finer spines completed the spiral. The
spines stand in a single row except in the
distal-most part where the edge of the penial
glans is broadened to a bulge (Figs. 6, 10,
12). All spines, the stylets of the ductus ejac-
ulatorius and the basal finger, and the thorn
at the opening through which the ductus
ejaculatorius leaves the penis are partly cov-
ered by an epidermal sheath (Figs. 7, 9, 10,
13), which, in the case of the spines on the
penial glans, bears bundles of presumably
sensory cilia (Fig. 9). The penis can be pro-
truded through a sheath (Figs. 1, 2, 13, 14),
which opens behind the right rhinophore.
The gonad of snail no. 1 contained only
spermatogonia and spermatozoa. Snails nos.
4 and 5 had in addition yolk material, but no
oocytes, whereas spermatognia, spermato-
zoa, yolk material and oocytes were found in
snails nos. 2 and 3. Specimen no. 3 had sig-
nificantly more oocytes than no. 2. In these
latter two individuals, the digestive gland
contained spermatozoa (Fig. 17). The am-
pulla of snail no. 2 had a distinct opening into
the digestive gland (Fig. 17). No such open-
ing was found in the remaining specimens.
This opening in snail no. 2 is no preparatorial
artefact as indicated by the extension of the
mass of sperm in the digestive gland far in
front of and behind the opening. A bundle of
spermatozoa lay in a fold of the foot close to
the anus of animal no. 3 (Fig. 18). These sper-
matozoa were obviously expelled through the
intestine at fixation. Spermatozoa were also
found in the posterior third of the visceral
hump in the kidney and in the haemocoel of
snail no. 2 (Fig. 19).
DISCUSSION
Acochlidium fijiense was described as her-
maphroditic (Haynes & Kenchington, 1991).
However, from the different states of gonadal
maturation we conclude that adolescent
gonochorism, that is beginning as a male (or
female, which does not apply in this case)
and then becoming a simultaneous her-
maphrodite (Ghiselin, 1987), may be a more
precise characterization of the reproductive
strategy of A. fijiense.
Our findings of the penial morphology dif-
fer in several aspects from the description of
Haynes & Kenchington (1991). These authors
observed neither the basal finger nor the true
course of the ductus ejaculatorius. Their
statement on number and position of the
spines on the edge of the penial glans varies
as well. These discrepancies are probably
ACOCHLIDIUM FIJIENSE 147
SO Е
FIGS. 13-15. Male genital organs. 13. Ductus ejaculatorius leaving penis; 14. Distal region of prostate and
paraprostate, penis and sperm conducting ducts. Arrows indicate spines, arrow heads the penial sheath;
15. Proximal region of prostate and paraprostate with ejaculatory duct. de = ductus ejaculatorius; dg =
digestive gland; oe = oesophagus; p = penis; ppr = paraprostate; pr = prostate; ps = penial sheath; sg =
salivary glands; th = thorn; vd = vas deferens. Scale bars = 100 um.
due to different states of contraction of the
penis after fixation. Besides, Haynes & Ken-
chington (1991) neither had the opportunity
of SEM investigations nor did they section
the penis.
For similar reasons, comparison with other
species of the genus Acochlidium are difficult.
What Bucking (1933) described as an oviduct
in A. amboinense Strubell, 1892, is clearly the
paraprostatic duct with the basal finger. And
in A. sutteri Wawra, 1979 (Wawra, 1979), the
smaller thorn appears to represent the arma-
ture of the basal finger or its homologue. The
descriptions of A. bayerfehlmanni Wawra,
1980 (Bayer & Fehlmann, 1960; Wawra, 1980)
and A. weberi (Bergh, 1896) (Bergh, 1896) are
too superficial to allow a detailed comparison.
Organs similar in structure and position to
the paraprostate and the basal finger of A.
fijense have been described in another aco-
chlidioidean species, the interstitial Pseu-
dunela cornuta (Challis, 1970) (Challis, 1970:
37). The penial gland and the ‘‘complex mus-
cular organ equipped with a single, hollow,
curved spine” of P. cornuta are probably re-
spective homologues.
The basal finger probably functions as a
stimulatory organ analogous to the dart (gyp-
sobelum) of some stylommatophoran land
snails or other organs in some other pulmo-
nates (Tompa, 1984). Adamo & Chase (1988,
1990) found that in Helix aspersa O. F. Muller,
1774, dart shooting decreased courtship du-
ration. In that species, the active substance is
secreted by the digitiform glands which pro-
duce a mucus that coats the dart. This mu-
cus is only effective if it is injected into the
body cavity (Adamo & Chase, 1990; Chung,
1986). We assume that in A. fijiense the basal
finger in association with the paraprostate
has a similar function during copulation.
The three strategies of sperm transfer oc-
curring in the order Acochlidioidea, namely
copulation, injection, and transfer by sper-
148 HAASE & WAWRA
FIGS. 16-19. Spermatozoa. 16. Gonad; 17. Ampulla opening into digestive gland; both organs are filled
with sperm; 18. Sperm in a fold of the foot. Arrow indicates the anus; 19. Sperm in haemocoel and kidney.
Arrows indicate blood cells; Figs. 16, 17 and 19 are interference contrast photographs. am = ampulla; c =
haemocoel; dg = digestive gland; dgd = distal gonoduct; go = gonad; in = intestine; k = kidney; pg =
praeampullary gonoducts; sp = spermatozoa; to = [presumptive (see Discussion)] temporary opening of the
ampulla into the digestive gland. Scale bars = 100 um.
matophores, are briefly discussed by Wawra
(1992). In A. fijiense, the penial armature,
the stylet-bearing ductus ejaculatorius and
the fact that we found spermatozoa in the
haemocoel and in the kidney of snail no. 2
indicate that sperm are injected into the
haemocoel at copulation. The injection of
sperm into the kidney was probably an acci-
dent.
Hypodermic impregnation is the presump-
tive mode of sperm transfer in another aco-
chlidioidean species, the interstitial Hedylop-
sis spiculifera (Kowalewsky, 1901) (Wawra,
1989). Hypodermic injection of sperm is fur-
ther known in some Sacoglossa (Baba & Ha-
matani, 1970; Gascoigne, 1956, 1975, 1976,
1978, 1993; Hand & Steinberg, 1955; Jen-
sen, 1986; Marcus, 1973; Reid, 1964; Trow-
bridge, 1995) and two nudibranch species
(Rivest, 1984). Gascoigne (1993) distin-
guished between precise and imprecise hy-
podermic injection. In the first, more common
ACOCHLIDIUM FIJIENSE 149
mode, sperm are injected through the body
wall directly into parts of the genital system,
while in the imprecise mode, spermatozoa are
released into the haemocoel. Imprecise hy-
podermic impregnation is only reported for
the sacoglossans Elysia maoria (Powell, 1937)
(Reid, 1964), E. subornata Verrill, 1901
(Jensen, 1986), Bosellia corinneae Marcus,
1973 (Marcus, 1973), and Alderja modesta
(Lovén, 1844) (Hand 8 Steinberg, 1955).
Hedylopsis spiculifera and our study organ-
ism, Acochlidium fijense, probably practice
the imprecise mode, too. In none of the im-
precisely injecting species is the fate of the
transferred sperm known. That holds also for
those acochlidioideans in which sperm trans-
ferred in spermatophores attached to the
body wall enter the haemocoel through lysis
of the recipient's epidermis (Doe, 1974; Had-
field & Switzer-Dunlap, 1984; Morse, 1976;
Swedmark, 1968a, b).
In the nudibranchs Palio zosterae (O’Dono-
ghue, 1924) and P. dubia (Sars, 1824), sperm
must be injected into the gonadal acini.
Sperm that are released into the haemocoel
are phagocytosed by blood cells (Rivest,
1984). In the specimen of A. fijiense impreg-
nated with sperm, we found accumulations
of blood cells, too. But because these sper-
matozoa are presumably intended to fertilize
eggs, the blood cells have probably a differ-
ent function such as nourishment or guid-
ance to the fertilization site.
Like most acochlidioideans, A. fijiense
lacks both a seminal receptacle for storage
of allosperm and a gametolytic gland (bursa
copulatrix) to digest excess allo- and auto-
sperm and other surplus substances and
products of the genital system (Hadfield 8
Switzer-Dunlap, 1984). Becausethese sperm-
receiving organs are typical of the Bauplan of
genital systems of opisthobranchs (e.g., Sal-
vini-Plawen, 1991), we consider their loss to
be secondary (see below). The loss of the
gametolytic gland appears to be compen-
sated by the digestive gland. In the five spec-
imens that we sectioned, only the ampulla of
snail no. 2 had an opening into the digestive
gland. Both this snail and individual no. 3 had
sperm in the digestive gland. Based on the
fact that ampulla and digestive gland were
connected in only one individual but two
snails had sperm in the digestive gland, and
supported by the consideration that both di-
gestion of food and release of gametes would
be hampered by a permanent opening of the
ampulla into the digestive gland, we conclude
that this connection is transient, established
only when required. Whether the digestive
gland also digests allosperm cannot be told.
This might be the case if sperm were acci-
dently injected into the digestive gland at cop-
ulation. If A. fijiense copulated through the
genital pore, sperm could reach the digestive
gland through the duct connecting distal
gonoduct and digestive gland. However, the
distal gonoduct has no vaginal characteristics
which would indicate reception of a copula-
tory organ and sperm. Circumstantial evi-
dence (penial armature, sperm in haemocoel
and kidney of snail no. 2) suggests that the
mode of sperm transfer is hypodermic injec-
tion.
Because the connection of ampulla and di-
gestive gland was seen in only a single indi-
vidual, one might argue that this opening was
an abnormality, and consequently, the sperm
in the digestive gland of individual no. 3
would be allosperm (see above). But the fact
that this opening was seen in only a single
snail is not a strong argument against the
presumed regularity of the temporary con-
nection of digestive gland and ampulla, sim-
ply because of the improbability to detect a
transient structure. Until further evidence we
intuitively prefer the first interpretation of the
findings in snail no. 2.
The loss of seminal receptacle and game-
tolytic gland in the genital system of acoch-
lidioidean species appears to be correlated
with the mode of sperm transfer. Strubellia
paradoxa is the only acochlidioidean species
possessing both organs (Wawra, 1988). Only
the gametolytic gland is present in Pseu-
dunela cornuta (Challis, 1970). In both spe-
cies, allosperm must enter the genital system
through the genital opening in order to get to
the receptacle or to the gametolytic gland. All
other species for which the genital anatomy
is described, including those of the genus
Hedylopsis Thiele, 1931 (contra Odhner,
1937, and Rankin, 1979, see Wawra, 1989),
have lost both the receptacle and game-
tolytic gland. In all these species, sperm are
or are presumed to be transferred either by
hypodermic impregnation or through sper-
matophores attached to the body wall of the
mating partner. All these species are either
small, interstitial forms or relatively large
freshwater snails of the genus Acochlidium
(not sensu Rankin, 1979; the species are
listed in Haynes & Kenchington, 1991). Be-
cause of the poor state of knowledge on the
majority of the species of the order Acochlid-
150 HAASE & WAWRA
ioidea, it is too early to speculate how often
these modes of sperm transfer accompanied
by the loss of the receptacle and the game-
tolytic gland evolved in this group. This holds
also for the question whether form and func-
tion of the genital system of Acochlidium are
autapomorphies of the genus or inherited
from a marine, possibly even interstitial, an-
cestor.
The bundle of sperm observed in a fold of
the foot near the anus of snail no. 3 was ob-
viously expelled from the intestine at fixation.
In this way, the animal decreased its volume
and thus could contract more efficiently. The
decrease of volume of the digestive gland
could in addition be achieved through the by-
pass to the distal gonoduct. However, the
true function of the duct connecting the di-
gestive gland and the distal gonoduct re-
mains a matter of speculation. A noteworthy
analogy exists in some turbellarians where
the ductus genitointestinalis connects the
genital system with the digestive system
(e.g., Reisinger, 1968). The true function of
this ductus genitointestinalis is also unclear.
The peculiar course of the vas deferens
was described earlier for A. sutteri (Wawra,
1979) and A. bayerfehlmanni (Wawra, 1980),
but it remained unclear through which duct
eggs are laid. In A. fijiense—and we assume
that the same holds for the above mentioned
species—the distal gonoduct continues be-
yond the branch to the vas deferens to open
anteriorly. It thus provides the passage for
the eggs to the exterior. Egg masses and lar-
vae of A. fijiense were described by Haynes &
Kenchington (1991).
Some of our conclusions as to the function
of the genital system of A. fijiense are neces-
sarily speculative. Observations of and ex-
perimentation with living animals have to
complement our findings and will confirm or
falsify our hypotheses.
ACKNOWLEDGEMENTS
We are greatful to Dr. H. Hilgers for sec-
tioning one specimen. Dr. H. Kothbauer, Dr.
B. Ruthensteiner, Dr. L. Salvini-Plawen and
two anonymous referees made helpful com-
ments on the manuscript.
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Revised Ms. accepted 20 February 1996
MALACOLOGIA, 1996, 38(1-2): 153-160
QUANTIFICATION OF THE DEVELOPMENT OF THE CEPHALIC SAC AND
PODOCYST IN THE TERRESTRIAL GASTROPOD LIMAX MAXIMUS L.
С. M. Kuchenmeister', D. J. Prior? & I. G. Welsford?
ABSTRACT
During embryonic development in the terrestrial gastropod Limax maximus L., an anterior
cephalic sac and a posterior podocyst are elaborated. At 4 + 1.2 d (X + SD; n = 25), rhythmic
contractions began in the podocyst. By 6.3 + 0.8 d of development, the cephalic sac began
rhythmic contractions in antiphase to those of the podocyst; a behavior we have termed
cephalopedal pumping. Pumping preceded regular heart activity, which began at 8 + 1.8 d of
development, and progressively decreased in frequency throughout embryonic development,
ceasing by hatching. Cephalopedal pumping was capable of redistribution of dye throughout
the embryo. The two structures were capable of sustained, independent, rhythmic contrac-
tions. Because pumping precedes regular heart activity and can, presumably, redistribute
hemolymph, cephalopedal pumping may serve as a primordial circulatory system, separate
from the developing cardiovascular system.
Key words: circulatory system, development, Limax maximus, slug, gastropod, heart
INTRODUCTION
Terrestrial gastropods such as slugs un-
dergo direct development and are easily
raised in the laboratory. Due to the combina-
tion of these characteristics, slugs drew early
attention as model developmental systems
(e.g., Laurent, 1837; Jourdain, 1884; Hench-
man, 1890; Cuenot, 1892; Kofoid, 1895; Car-
dot, 1924). lt has been known for over 100
years that slug embryos elaborate an anterior
cephalic sac (= anterior vesicle, Laurent,
1837; Jourdain, 1884; = vesicle, Kofoid,
1898; = ectodermal sac, Simpson, 1901) and
a posterior podocyst during development. It
has also been widely reported that, once
formed, these structures undergo rhythmic
contractions. It has been proposed that the
rhythmic contractions serve а circulatory
function in embryos (e.g., Jourdain, 1884). Al-
ternatively, the contractions of the cephalic
sac and podocyst have been suggested to
control movement of the embryo within the
egg (Laurent, 1837; Jourdain, 1884), a circu-
latory function (e.g., Cuenot, 1892; Cardot,
1924) and/or serve a respiratory or osmoreg-
ulatory function (Laurent, 1837; Jourdain,
1884; Kofoid, 1895).
Although the development of the podocyst
and cephalic sac has been used as a quali-
tative component of a staging scheme for
some species of slugs (Carrick, 1938), the
development of these structures has yet to
be quantified. Furthermore, there exists very
little data on the potential physiological sig-
nificance of these structures and/or their
contractile behavior. We have initiated inves-
tigations into the development of the cepha-
lic sac and podocyst of the terrestrial slug
Limax maximus L. and compared this with
the development of heart in an attempt to
quantify both the morphology and contractile
behavior of these structures during develop-
ment. A preliminary report of these data has
appeared in abstract form (Welsford 8 Prior,
1988).
MATERIALS AND METHODS
Animals
Sexually mature (according to the criteria
of Sokolove £ McCrone, 1978) L. maximus,
which had either been collected from the field
or raised from eggs in a laboratory culture,
were kept in vented containers lined with wa-
ter-saturated paper towels under a regulated
light cycle (L:D 14:10 or 13:11), a regulated
temperature cycle (18°C during lights on and
"Department of Biology, Bradley University, Peoria, Illinois 61625, U.S.A.
“Northern Michigan University, Marquette, Michigan 49855, U.S.A.
“Department of Biology, Keene State College, Keene, New Hampshire 03435-2001, U.S.A., to whom correspondence
should be addressed.
154 KUCHENMEISTER, PRIOR & WELSFORD
12°C during lights off) and approximately
100% RH. Slugs were fed ad libitum on lab-
oratory food pellets (Purina Rat Chow). Limax
maximus lays its eggs in discrete clumps or
masses of 20-250 eggs, thus facilitating
identification of eggs laid by an individual an-
imal (Prior, 1983). Egg masses were collected
daily from containers and placed on water-
saturated filter paper-lined petri dishes at a
constant temperature (either 15°C or 20°C)
under a light cycle (L:D 14:10).
Morphological Measurements
Every 2 d, embryos were decapsulated
(i.e., removed from the eggs) in a dish of ster-
ile slug saline (55.6 mM Ма*, 4.2 mM К*, 7
mM Са**, 4.6 mM Mg'**, 80.3 mM СГ, 0.2
mM H,PO, , 5.0 mM НСО., 5.0 mM Dex-
trose, pH 7.3-7.41, 139-145 mOsm/kg H,0;
Prior 4 Grega, 1982). Decapsulated embryos
were transferred by pipette to a depression
slide and viewed under a compound micro-
scope fitted with an ocular micrometer (Nikon
Optiphot Il or Olympus Bmax). The follewing
measurements were taken (in um): (1) maxi-
mal width of the cephalic sac; (2) maximal
length of the cephalic sac; (3) maximal length
of the foot; (4) maximal length of the
podocyst; (5) maximal width of the podocyst;
(6) maximal width of the pericardial chamber
(Fig. 1A,B). To allow for comparisons of rel-
ative structural dimensions at varying embry-
onic stages, all measurements were normal-
ized to the length of the foot because this
structure is readily identifiable at all develop-
mental stages.
To determine whether cephalopedal con-
tractions could distribute dye throughout the
embryo, a microcapillary tube was filled with
2% Blue Dextran (MW approximately 2 X 10°:
Sigma Chemical Co., Inc.) and inserted into
either the cephalic sac or podocyst with the
aid of a micromanipulator (Leitz or WPI inc.).
Dye was injected using low pressure (not
greater than 0.5 atm). To control for potential
damage during injection, only data from em-
bryos which exhibited normal contraction ac-
tivity for at least 20 min after injection were
reported. To more clearly delineate the re-
gions throughout which contractions were
distributing dye, a saturated carmine solution
was injected into either the cephalic sac or
podocyst. Following injection with carmine,
embryos were placed in a saturated solution
of chlorobutanol for 30 min to ensure mus-
cular relaxation (Kempf, personal communi-
cation), fixed in paraformaldehyde, dehy-
drated, embedded in paraffin, sectioned at
10 um thickness and mounted onto slides.
Sections were stained with Harris’ Hematox-
ylin and counter stained with Eosin Y follow-
ing standard protocols (Schleicher, 1953).
Stained sections were observed under light
microscopy and the distribution of carmine
particles was determined. Sections were
drawn with the aid of a drawing tube attach-
ment to a Nikon Optiphot II.
Physiological Measurements
To ensure that decapsulation had no effect
on the contractions of the cephalic sac,
podocyst or (in later stages) heart, the rate of
heart and/or cephalopedal contractions was
determined prior to and following decapsula-
tion. Contractile activity was measured by
placing the embryo on a modified depression
slide which allowed the embryo to be contin-
uously superfused with saline. Embryo be-
havior was recorded on VHS videotape using
a videomicroscope (Zeiss or Olympus).
Statistical Analysis
Comparisons between developmental
times at varying temperatures and between
heart rate and cephalopedal contraction rate
were performed using a T-test for indepen-
dent samples. Trends for heart and cephalo-
pedal contraction during development were
determined by calculating linear regressions.
Statistical analyses were performed using
either PsiPlot (PsiPlot Inc.) or DataDesk (Data-
Desk Inc.), and graphs were constructed
using either PsiPlot or Kaleidograph (Kalei-
dograph Inc.). In all comparisons, a probabil-
ity values of less than 0.05 was considered
significant.
RESULTS
Development of the Cephalic Sac,
Podocyst and Heart
Although there was considerable variation
in the developmental time of different animals
within a clutch and between clutches, embry-
onic development in L. maximus averaged
26.6 + 13.6 X + SD; п = 15 clutches) dat
20°C. Development was temperature-depen-
dent, taking significantly longer at 15°C (35.6
+ 19.2 d; t = 4.97, p = 0.00011). The cephalic
CEPHALOPEDAL PUMPING IN L. MAXIMUS
A
— Cephalic Sac
ie Tentacles
De Mouth
Podocyst ——
100 um
1
B
2
1= Maximum Width of Cephalic Sac
2= Maximum Length of Cephalic Sac
3= Maximum Length of Foot
4= Maximum Length of Podocyst
5= Maximum Width of Podocyst
6= Maximum Width of Pericardium
155
FIG. 1. A: Drawing of a slug embryo at 15 d of development indicating the relative positions of the podocyst,
foot, cephalic sac and pericardial area. B: Indicates the morphological measurements taken at each
embryonic stage. Scale bar is 100 um.
156 KUCHENMEISTER, PRIOR & WELSFORD
sac developed within 4 d of egg laying and
decreased progressively in size relative to the
foot throughout embryonic development (Fig.
2A). The cephalic sac invaginated within
22-26 d of development and, consistent with
the observations of Carrick (1938) for Agrioli-
max agrestis, was observed to form compo-
nents of the internal organs including the al-
bumin glands and reproductive structures.
The sac was absent by hatching in all animals
observed (n = 350).
The podocyst also developed within the
first 4 d, and increased in size relative to the
foot throughout the first 20-22 d of develop-
ment. The podocyst decreased in size after
this time, usually disappearing prior to hatch-
ing (Fig. 2B). Occasionally a hatchling slug
retained a remnant of the podocyst after
hatching, but this was lost in all observed
cases within 3 d post-hatch (n = 350).
The heart first appeared between 10-12 d
of development from a dorsal evagination of
the embryo just caudal to the cephalic sac.
The heart increased in size relative to the foot
until approximately 18 d of development then
decreased in relative size thereafter (Fig. 2C).
Cephalopedal Contractions
By 4 + 1.2 (X + SD; n = 25) d of develop-
ment, the podocyst initiated irregular, low fre-
quency contractions (16.2 + 5.4 beats per
minute [BPM]; X + SEM; n = 45; Fig. 3A). By
day 5 (56.4 + 1.8 d), the cephalic sac began
contractions in antiphase to the podocyst.
Cephalopedal contractions peaked in fre-
quency at approximately 4 d of development
and decreased in frequency throughout de-
velopment (r = —0.932; р < 0.0001; Fig. ЗА).
After 18 d of development, there was а
marked increase in the variance of the
cephalopedal contractions (Fig. 3A.).
The rate of cephalopedal contractions was
unaffected by decapsulation (Table 1). The
antiphasic nature of the cephalopedal con-
tractions led us to investigate the nature of the
oscillatory control of contractions. The con-
tractions of both the podocyst and cephalic
sac were unaffected by rupture or removal of
the other structure (Table 1) or, indeed, by
complete transection of the embryo, with the
exception that the structures ceased to con-
tract in antiphase to one another (Table 1). The
independent contractions in each structure in
completely transected embryos lasted for an
average of 2 h (2 + 8.7 h; n = 15) and in one
instance, continued for 20 h after transection.
A. Cephalic Sac Development
4
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С. Heart Development
Normalized Measures
0 2 4 6 8 10121416182022242628
Days of Development
FIG. 2. Means of normalized measures of cephalic
sac width (A), podocyst width (B) and pericardial
area width (C) during embryonic development.
Each bar represents the mean value of 55 animals.
CEPHALOPEDAL PUMPING IN L. MAXIMUS 197
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Days of Development
FIG. 3. A: Frequency (in BPM) of cephalopedal
pumping in embryos of varying ages (measured at
20°C). Each point represents the mean (+ SD) re-
sponse of 45 animals. B: Frequency (in BPM) of
heart activity in embryos of varying ages. Each
point represents the mean (+ SD) response of 45
animals.
Heart Contractions
In contrast to the cephalopedal activity,
heart activity did not begin until 9.2 + 1.4 d of
development (Fig. 3B). Heart rate was signif-
icantly greater than cephalopedal contrac-
tions at all stages (mean across all stages for
heart was 57.4 + 156.7 BPM vs. 7.1 + 25.7
BPM; t = 6.545; p < 0.0001). Heart rate
peaked at 9 d of development and decreased
progressively throughout development (r° =
0.7; p < 0.0001), but with a significantly dif-
ferent slope than that of cephalopedal con-
tractions (-2.0 BPM/d for heart vs. —0.64
BPM/d for cephalopedal contractions; Fig.
3A,B). Initially, heart rate was strongly cou-
pled to pumping activity, cyclically increas-
ing and decreasing with each cephalopedal
contraction (Fig. 5A). After 14 d of develop-
ment, heant activity became independent of
cephalopedal activity (Fig. 5B). Heart activity
was highly variable throughout the first 18 d
of development, but the variance of heart ac-
tivity decreased from 18-20 d of develop-
ment to hatching (Fig. 3B).
Staining of Intra-Embryonic Regions
Cephalopedal contractions were capable
of redistributing dye throughout the embryo
(Fig. 6). However, the portion of the embryo
that was stained by this procedure depended
upon the age of the embryo. Injection into the
podocyst in embryos younger than 10 d, re-
sulted in staining of primarily the entire pos-
terior region of the embryo, including a region
surrounding the CNS and heart (summarized
in Fig. 6). The cephalic sac was stained by
podocyst injections only lightly and only after
prolonged injections (>5 min). Injections into
the cephalic sac in embryos younger than 10
d resulted primarily in staining a restricted
internal space distinct from that stained with
the podocyst injection and continuous with
the stomach, reproductive organs and he-
patopancrease (Fig. 6). Injection into the
cephalic sac only lightly stained the podocyst
and heart and only after prolonged injections.
In embryos older than 12 d, injection into the
podocyst or cephalic sac did not stain the
heart. In addition, in embryos older than 12 d,
injections into the podocyst in excess of 10
min failed to stain the regions of the embryo
continuous with the cephalic sac (Fig. 6). At
all stages of embryonic development tested,
histological sections confirmed the dye sep-
aration of the internal regions continuous
with either sac or podocyst in embryos older
than 12 d (Fig. 6).
DISCUSSION
The observations in the present study are
consistent with qualitative observations on
the development of Agriolimax agrestis (by
Carrick, 1938) and Limax maximus (by Simp-
son, 1901) and thus support the utility of the
use of Carrick's (1938) scheme (developed
for use with embryos of A. agrestis) for the
staging of embryos of Limax maximus. Be-
cause preliminary observations suggest that
this scheme also holds for the slugs Lehma-
nia valentiana and Agriolimax (=Deroceros)
reticulatus, this scheme may be generalizable
to all slug species. The availability of a stag-
ing scheme for slugs may aide in the approx-
imate aging of embryos for which the date of
158 KUCHENMEISTER, PRIOR & WELSFORD
TABLE 1. The effect of rupture or removal of either the cephalic sac or podocyst on the rate of cephalic
sac, podocyst and heart contractions is shown, In addition, the effect of complete embryo transection
on cephalic sac and podocyst contractions is shown. Data are mean (+ SD) response of 15 animals.
Rate in Egg Case
Structure (BPM) (BPM)
Podocyst 25 + 12.6 23 + 13 (91%)
Cephalic Sac РЗ 14-3 22 + 18 (98%)
Heart 134 + 23 122 + 28 (91%)
A. Heart Activity at 10d of Development
140
120
100
Heart Rate (BPM)
Relaxed Contracted
Podocyst Contraction Cycle
B. Heart Activity at 20d of Development
100
80
60
40
Heart Rate (BPM)
20
Contracted
Relaxed
Podocyst Contraction Cycle
FIG. 4. Differences in sensitivity of heart activity to
cephalopedal activity at 10 d of development (A)
and 22 d of development (B). In each figure, the
mean (+ SD) instantaneous heart rate is shown (ex-
pressed in BPM) during podocyst contraction and
relaxation. Each bar represents the mean (+ SD)
response of 45 animals.
laying of a clutch is unknown and/or which
have developed under variable thermal con-
ditions, because the development is mark-
edly temperature sensitive.
The cephalic sac and podocyst developed
earlier than the heart and began rhythmic,
Rate in Saline
Rate 60 min Post-
Rupture of Opposing
Structure (BPM)
22 + 14 (96%)
19 + 17 (86%)
119 + 47 (98%)
Rate 60 min Post-
Transection (BPM)
20 + 16 (97%)
21+19(110%)
121 +59 (101%)
antiphasic contractions prior to the onset of
heart activity. The frequency of cephalopedal
contractions was significantly lower than that
of heart and the rate of decrement in activity
throughout development between heart and
cephalopedal contractions differed signifi-
cantly. In addition, heart and cephalopedal
contractions demonstrated differing patterns
of variance, with heart becoming more regu-
lar as cephalopedal contractions became
less regular. Because podocyst and cephalic
sac contractions were capable of redistribut-
ing dye throughout the embryo, contractions
of these structures could accomplish the cir-
culation of hemolymph throughout various
regions of the embryo prior to maturation of
the cardiovascular system. We have thus
termed the rhythmic antiphasic contractions
of the cephalic sac and podocyst, cephalo-
pedal pumping.
The fact that heart activity was significantly
affected by cephalopedal pumping at early
stages suggests that, early in development, it
may be continuous with the cephalic sac and
podocyst, but that this connection is lost as
the embryo matures. This hypothesis was
supported by dye injections into the cephalic
sac and podocyst, which stained the lumen
of the heart in early stages, but not in later
stages, of L. maximus embryos.
Both the cephalic sac and the podocyst
can contract independently of one another.
Thus, each structure may be driven be sep-
arate oscillators that are coupled to one an-
other. Because the CNS reportedly develops
within approximately 9 d in embryos of L.
maximus (Henchman, 1890), the nature of
such putative oscillators remains uncertain
as does their fate in hatchling slugs. Work on
these oscillators is ongoing.
Gastropod mollusks have served as useful
model systems for the study of the develop-
ment of the central nervous system and be-
havior due to the relatively small number of
neurons present within the CNS and the fact
CEPHALOPEDAL PUMPING IN L. MAXIMUS 159
Cephalic sac
A
— Podocyst
100 um
Cephalic sac
\
B — Podocyst
Shell
Cephalic sac / ART
e
—— Podocyst
100 um
FIG. 5. Drawings of histological sections of embryos at 5 d of development (A), 15 d of development (B) and
25 d of development (C). In each figure, the stippled areas denote the regions stained by injection of
Carmine solution into the podocyst (A and В) or cephalic sac (С). Scale bar is equal to 100 um. STM =
N ALB = albumin gland, BM = buccal mass, CNS = central nervous system, TENT = tentacles, HRT
= heart.
160 KUCHENMEISTER, PRIOR & WELSFORD
that neurons are frequently large and easily
identifiable from individual to individual.
However, considerably less information is
available on the development of peripheral
structures in these organisms. Investigations
of the development of the peripheral struc-
tures are hampered in many mollusks by a
metamorphosis that includes a veliger stage
and can entail dramatic alterations in central
and peripheral morphology and physiology.
Furthermore, the housing and maintenance
of these organisms frequently requires mari-
culture facilities. Terrestrial gastropods are
easily raised the laboratory and thus may
serve as useful model systems for certain de-
velopmental investigations.
ACKNOWLEDGMENTS
This work was supported in part by the
OTEFD at Bradley University, a Whitehall
Foundation Grant to IGW an MBRS grant to
DJP and NSF DUE—935273 (IGW). The au-
thors wish to thank RR Stephens for com-
ments on the manuscript and Drs. G. E. Gos-
low and D. Blinn for the kind use of facilities
during the initial stages of these investiga-
tions.
LITERATURE CITED
CARRICK, R., 1938, The life history and develop-
ment of Agriolimax agrestis L. the gray field slug.
Transactions of the Royal Society of Edinburgh,
59 (Part 3)(21):563-597.
CARDOT, H., 1924, Observations physiologiques
sur les embryons des gasteropodes pulmones.
Journal de Physiologic et de Pathologie Gen-
erale, 22:575-586.
CUENOT, L., 1892, Etudes physiologiques sur les
gasteropodes pulmones. Archives de Biologie,
12:683-740.
HENCHMAN, A. P., 1890, The origin and develop-
ment of the central nervous system in Limax
maximus. Bulletin of the Museum of Compara-
tive Zoology (Harvard University), 20:169-209.
JOURDAIN, M. S., 1884, Sur les organes segmen-
tares et le podocyste des embryones de Lima-
ciens. Comptes Rendus Hebdomadaires des Sé-
ances de l'Academie des Sciences, 97:308-310.
KOFOID, C. A., 1895, On the early development of
Limax. Bulletin of the Museum of Comparative
Zoology (Harvard University), 27:35-135.
LAURENT, M. 1837, Observations sur le developp-
ment des oeufs de la limace grise et de la limace
rouge. Comptes Rendus Hebdomadaires des
Séances de l’Academie des Sciences, 4:295-
297.
PRIOR, D. J. 1983, The relationship between age
and body size of individuals in isolated clutches
of the terrestrial slug, Limax maximus (Linnaeus,
1758). Journal of Experimental Zoology, 225:
321-324.
PRIOR, D. J. & D. S. GREGA, 1982, Effects of tem-
perature on the endogenous activity and synap-
tic interactions of the salivary burster neurones
in the terrestrial slug, Limax maximus. Journal of
Experimental Biology, 98:415-428.
SCHLEICHER, Е. M., 1953, An improved hematox-
ylin-eosin method for sections of bone marrow.
Stain Technology 28:119-123.
SIMPSON, G. B., 1901, The anatomy and physiol-
ogy of Polygyra albolabris and Limax maximus,
and the embryology of Limax maximus. Bulletin
of the American Museum of Natural History,
8:237-311.
SOKOLOVE, P. G. & E. J. MCCRONE, 1978, Re-
productive maturation of the slug, Limax maxi-
mus, and the effects of artificial photoperiod.
Journal of Comparative Physiology, 125:317-
325.
WELSFORD, I. С. & D. J. PRIOR, 1988, Embryonic
cephalo-pedal pumping in the terrestrial slug,
Limax maximus. American Zoologist, 28:25A.
Revised Ms. accepted 8 April 1996
MALACOLOGIA, 1996, 38(1-2): 161-180
LOCAL PATTERNS OF LAND SNAIL DIVERSITY IN A KENYAN RAIN FOREST
P. Tattersfield
Bettfield Clough Cottage, Castleton Road, Chapel-en-le-Frith Stockport SK12 6PE,
United Kingdom
ABSTRACT
Terrestrial molluscs were sampled in indigenous forest and plantation plots in Kakamega
Forest, western Kenya, which is the eastern-most patch of Guineo-Congolian rain forest in
Africa. Fifty species (one slug and 49 snails) were recorded from 27 indigenous forest plots, and
the mean species per plot was 23.4. The majority of the species present in the fauna were small,
litter dwellers, with 52% having a major shell dimension of less than 5 mm. Overall, species
richness and faunal composition were relatively uniform throughout the forest system. How-
ever, forest edge plots, including plots located along large rivers and in smaller blocks of forest,
had a deficiency of some minute, litter-dwelling species but supported a higher frequency of
some large-shelled taxa. The four plantations sampled supported fewer species per plot (15.25
species/plot) and also lacked several of the small, litter-dwelling species found in the indige-
nous forest.
Many other species of mollusc have been previously reported from Kakamega Forest. The
reported mollusc fauna of Kakamega Forest represents about 5.8-9.5% of the total known East
African forest mollusc fauna, thus suggesting that there must be considerable taxonomic re-
placement of species throughout the region. The recorded molluscan diversity in Kakamega
Forest is high in a worldwide context. Kakamega Forest is not old in geological terms, the Lake
Victoria basin having received a much more arid climate during periods of extended glaciation
at higher latitudes. Its forest fauna must have colonised since the last glacial maximum in Africa,
approximately 14000 years BP; the composition of the recorded fauna supports the view that
recolonisation was mainly from forest refugia in central Africa. The conservation implications of
the findings are discussed.
Key words: land snails, Gastropoda, biodiversity, rain forest, Africa, Kenya, Kakamega
Forest.
INTRODUCTION
Comparisons of land snail diversity at a re-
gional scale indicate that there are sometimes
large differences between tropical and tem-
perate zones (Cameron, 1995). At the local
scale, faunas from temperate sites are often
rich in species but quite uniform over large
geographical areas. However, there is little
comparable information about local diversity
patterns in tropical areas. Solem (1984) pro-
posed a model to account for world-wide land
snail diversity levels, but noted that much fur-
ther information is required about levels of
sympatric diversity in many parts ofthe world,
especially in the tropics. This lack of informa-
tion clearly has implications when trying to
assess the impact of habitat loss on mollus-
can biodiversity, and consequently for con-
servation planning (Cameron, 1995).
Much has been written about the East Af-
rica terrestrial mollusc fauna, but there have
been few investigations on molluscan as-
161
semblages in different habitat types. This
study investigates the patterns of land snail
diversity in a relatively restricted area of rain
forest habitat in western Kenya. It examines
areas of forest that have been subjected to
varying levels of human disturbance and ex-
ploitation and also surveys the mollusc fauna
in plantations of both exotic and indigenous
tree species.
THE SITE
The Kakamega Forest complex (about
0°15’N, 34°54’E) (Fig. 1) lies mostly to the
west of Kakamega town in west Kenya,
about 40 km north of Lake Victoria. It com-
prises several separate blocks of forest (Mu-
пик! 8 Tsingalia, 1990), of which Kakamega
Forest itself is by far the largest; the smaller,
isolated areas of Kisere and Mlaba forests lie
to the north of the main forest block. Bunyala,
Maragoli and Kaimosi forests are situated to
the northwest, south and southwest of Kaka-
162
SUDAN
ETHIOPIA
(| 5
\\
NN
\)
SOMALIA
mKitale KENYA
$ KAKAMEGA FOREST
Pkisumu AMtKenya
№ Nairobi
Kilimanjaro x
TANZANIA
N
;
o 100 200km
CHE
KEY |
o 7
Indigenous forest plot
Plantation plot
?
4
Road or track e
(
No 277
= Shinyaluÿ
Forest boundary /
\
м El
Village D D TRS
TATTERSFIELD
MLABA
\ FOREST
SR «
\
\
\
\
|
|
1
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y jf FOREST
/
/ D
aN
| e3 28
f 7
| ie LAN
+
KAKAMEGA
FOREST
FIG. 1. The location of Kakamega Forest and the sampling plots.
mega Forest, respectively. The area has a
relatively flat or gently undulating topogra-
phy at an altitude of approximately 1500-
1680 m. Two major rivers, the Yala and Isi-
ukhu, as well as many smaller watercourses,
run through the forest.
The forest complex covers an area of ap-
proximately 265 km”. About 45 km? is pro-
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 163
tected for wildlife as national reserve and na-
ture reserve, and some of the area is currently
proposed as a national park. The remainder is
gazetted as forest reserve and is managed
by the Forestry Department; current policy is
to encourage indigenous forest rather than
plantation. According to Muriuki & Tsingalia
(1990), about 48% of the forest complex sup-
ports indigenous forest stands, the remainder
containing plantations and grassland clear-
ings of both natural and anthropogenic origin.
Parts of the forest were selectively logged
in the 1930s, 1940s, late 1970s and early
1980s (Tsingalia, 1990). The region contains
some of the densest population in Kenya, and
until a presidential decree in 1986, many parts
of the forest were subject to shifting cultiva-
tion by local people. Kokwaro (1984) noted a
rapid decline in the extent of indigenous for-
est. Such activities have now been stopped,
but other forms of illegal exploitation still take
place, especially the removal of plant prod-
ucts for medicinal use and firewood, stock
grazing and poaching for small game. If
caught, offenders face a stiff punishment,
with fines for a first offence equivalent to one
month’s income or three months in prison.
The climate can be described as relatively
hot and humid, although rainfall is seasonal,
with most falling between March and July
and again in October and November. Muriuki
& Tsingalia (1990) report mean annual rainfall
as 2216 mm, although Zimmerman (1972) re-
corded over 3500 mm during 1963, indicat-
ing substantial fluctuation from year to year;
mean monthly maximum temperature ranges
from 18-29°C (Muriuki & Tsingalia, 1990).
The site is biogeographically important,
being situated at the edge of several regional
vegetation zones. White (1983) classifies it as
transitional rain forest, noting that it supports
several Guineo-Congolian plant species at
their easternmost African limit. The fauna
also has strong central and west African af-
finities (Faden, 1970; Zimmerman, 1972). Lu-
cas (1968) describes Kakamega Forest as
“the most easterly point of the West African-
Congo type forest.” It supports many plant
and animal species which are not found else-
where in Kenya (Faden, 1970).
METHODS
Plot Selection and Description
Thirty one plots (1-31), each approximately
40 m x 40 m, were sampled in Kakamega,
Kisere and Mlaba forests; 27 of the plots
were in mixed, indigenous forest, the other
four being in plantations (Fig. 1). Maximum
plot separation was approximately 30 km.
Forest edges were avoided during selection
although three plots were situated at the
edge of the Yala and Isiukhu rivers (Plots 6, 7,
22) and contained marginal, riverine forest
vegetation locally dominated by light de-
manding tall herbaceous species; plot 23 lay
within about 50 m of the Yala River but did
not contain forest edge.
Twenty three of the indigenous forest plots
contained mature, mixed stands (canopy
height generally about 20-30 m). All had been
exploited for wood, timber or other resources
to some degree, although observations on
forest structure and other signs, such as the
presence of saw pits, freshly cut wood and
evidence of cattle grazing indicated that five
of these plots (Plots 10, 11, 14, 16 and 29)
were substantially more disturbed than the
others. The other four indigenous forest plots
(5, 9, 12, 30) contained relatively young,
mixed, stands. These plots were all charac-
terised by a relatively low canopy (about
10-20 m), high light penetration at ground
level, and a mixed and generally well-devel-
oped grassy herbaceous field layer. Some of
them contained Acanthus arborea Forsskal, a
characteristic plant species of disturbed sites
requiring high illumination. Details of former
land use are unknown, but it is possible that
these areas had been cut and subsequently
regrown or been recolonised by indigenous
forest.
All four plantations (Plots 20, 25, 26 and
31) were probably about 20-40 years old
and all had a well-developed canopy. The
trees were well spaced and had presumably
been thinned. Two contained monoculture
stands of the non-indigenous Bischofia jav-
anica Blume, one had been planted with
mixed, non-indigenous conifers (Pinus patula
Schlecht & Chamisso and Cupressus lusitan-
ica Mill.) and the other contained a monocul-
ture of the indigenous, Guineo-Congolian
lowland rain forest tree species Maesopsis
eminii Engl. The detailed history of these ar-
eas is not known, although it is probable that
they were formerly covered in indigenous for-
est, possibly with a period of cultivation prior
to conversion to plantation.
Physical plot characteristics were ге-
corded including topography, inclination, as-
pect, and presence and relative abundance
of such potential molluscan microhabitats as
dead wood, fallen trees, and rocks. Forest
164 TATTERSFIELD
structure was described by estimating can-
opy height and the percentage cover of dif-
ferent vegetation strata (tall, medium and low
tree components and scrub, herb and liane
categories) for each site. Many of the indig-
enous plots had a dense understorey shrub
layer of Dracaena afromontana Mildbr. Plant
species were not generally recorded, al-
though notes were made on common domi-
nant species where they could be identified.
Mutangah et al. (1992) provide further infor-
mation about the vegetation of Kakamega
Forest.
Mollusc Sampling
Sampling for molluscs was undertaken by
a combination of direct search and litter siev-
ing methods. Each plot was searched for at
least 30 minutes, ensuring that all potential
microhabitats, such as dead wood, rocks,
tree trunks, leaf litter and living vegetation,
were examined. It was not possible to survey
the forest canopy directly, but fallen trees
and canopy branches supporting epiphytic
orchids, mosses and lichens were examined
when available. All molluscs found were col-
lected. Up to three local guides assisted with
the direct searching and therefore sampling
effort varied amongst the plots. However, the
mean number of species per plot does not
differ significantly between the sites sampled
by one or three people (F, 23 a+ = 3.70, P >
0.05), and thus this variation in sampling ef-
fort does not appear to affect the assessment
of diversity levels. About 4 litres of surface
leaf litter and soil were taken from each plot
and passed through a coarse sieve (4 mm
mesh size). Large species retained in the
sieve were removed. The fine fraction was
then dried and passed through two further
sieves (mesh sizes 2 mm and 0.5 mm). These
sieve fractions were searched separately un-
der good illumination until no further mol-
luscs could be found (generally about 30-45
minutes). Material passing through the 0.5
mm mesh for the first few sites was searched
for snails, but because none were found, this
fraction was discarded for subsequent sam-
ples. Some specimens were preserved in
70% ethanol, the others were stored dry.
These sampling methods are similar to those
used in other studies (e.g., De Winter, 1995).
Identification and Analysis
Most molluscs have been identified to spe-
cies level and nomenclature is provided in
Appendix 1. A reference collection has been
sent to the National Museums of Kenya,
Nairobi, and the remainder of the material will
be deposited in the National Museum of
Wales, Cardiff (NMW Z 1993.062). Several
species in the urocyclid genus Thapsia are
present in the samples. Two of these (Thap-
sia microleuca Verdcourt and T. eucosmia
Pilsbry) are distinctive, but the other species,
of which there are at least two, are difficult to
separate and have been aggregated for the
purposes of analysis. Further notes on iden-
tification are provided in Appendix 1.
The number of individuals (separated into
living and dead specimens based on the
presence of body tissues and shell condition)
has been recorded for each of the samples.
However, since sampling effort varied
amongst the plots, it does not provide a mea-
sure of absolute species abundance or allow
direct comparisons between the plots. Most
of the analyses have therefore been based on
presence and absence data. Nevertheless,
the number of individuals does represent a
measure of the relative abundance of species
in the fauna and is therefore of some interest.
The analysis of diversity patterns has fol-
lowed Cameron’s (1992) methods. Two mea-
sures have been adopted, Whittaker’s (1975)
Index (/), the ratio of overall species number
(S) to the mean number of species per plot
(<), provides a measure of between plot
differences. An index of 1 reveals identical
faunas, whereas higher values demonstrate
increasing differentiation. High values of / can
either result from the geographical replace-
ment of taxa within the same habitat or along
habitat gradients (Cody, 1986). Following
Cameron’s (1992) methods, these effects
have been examined by calculating the ratio
of the variance of the number of sites per
species to the maximum variance possible
for the same values of $ and «<. Where re-
placement effects are important, as opposed
to random effects due to sampling error, the
achieved variance is low compared with the
maximum possible.
RESULTS
Table 1 lists the 53 species recorded from
the 31 plots during the survey. These consist
of one slug and 52 snail species. Seven spe-
cies were recorded only as dead shells and
one of these, Cecilioides species, has been
excluded from the analyses because it was
only found on Plot 7, which lies adjacent to
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 165
TABLE 1. Species of molluscs recorded in the 31 sampling plots in Kakamega Forest.
Species WI 5 6 7 В 9 1011 12 13 1415 16 № 18
Elgonocyclus koptaweliensis + +
Maizania elatior CET a Se pe
Succinea Sp. + + + at
Truncatellina ninagongonis + + + + + + + + +
Nesopupa bisulcata + + + + PUNTO ne
Pupisoma harpula + + + + + + +
Pupisoma orcula + + ae Wap
Pupisoma sp. A + + E + re
Pupisoma sp. B + + + + ep
Acanthinula sp. + +
Rhachidina chiradzuluensis var. virginea + +
Conulinus rutshuruensis major + + Frei
Cerastua trapezoidea lagariensis + + + +
Micractaeon koptawelilensis + + + + + + + + + + + + + + + +
Nothapalus sp.
Subulona clara + + + + +
Oreohomorus iredalei 4. m
Pseudoglessula elegans + + + + + + + + + + + + + + + +
Pseudopeas cf. yalaensis + + + + + + + + + + + + + + + +
Curvella sp. A + + + + + + + + + + + +
Curvella cf. babaulti + + + + + + + + + + + + + + +
Achatina stuhlmanni + + +
Limicolaria cf. saturata
Punctum ugandanum + + + + + + + + + +
Punctum sp. A = + + + + + + + + dE
Punctum sp. B - + + + + +
Trachycystis iredalei + + + + + + + + + + + +
Trachycystis ariel $ + ap
Prositala butumbiana - + + + + + + + +
Kaliella barrakporensis + + + + + + + + + + + + + + +
Kaliella iredalei + + + + + + + + + + + + + + + + +
carinate species + + + + + + + + + + + + + + + + + +
Guppya quadrisculpta + + + + + + + + + + + + + + + + + +
Afroconulus iredalei + + + + + +
Trochozonites cf. medjensis + + +
Thapsia eucosmia + + +
Thapsia тисгоеиса + + - - + + + + +
Thapsia spp. + à + E EE Ot Fo He € À ‘++ +
Gymnarion aloysiisabaudiae + + + + + + + + + + + + +
Chlamydarion oscitans + + + + +
urocyclid slug
Halolimnohelix percivali
Halolimnohelix plana + + + 4:
Gonaxis elgonensis + + + + + + +
Gulella woodhousei + + + + + + + + + + +
Gulella osborni + + + + + + + + + + + + + + + +
Gulella impedita + + + + a hy E + + + Pr
Gulella ugandensis sE + + +
Gulella lessensis + + + + + + + + +
Gulella handeiensis +
Gulella disseminata + + + + + + + ++ +4 + + + + + + + +
Streptostele bacillum + + + + + + + + + + + + + +
Cecilioides sp. +
Site totals 30 22 24 20 19 33 27 27 22 24 30 18 19 19 23 20 23 24
(continued)
166
TATTERSFIELD
TABLE 1. Species of molluscs recorded in the 31 sampling plots in Kakamega Forest. (continued)
Species
Elgonocyclus koptaweliensis
Maizania elatior
Succinea sp.
Truncatellina ninagongonis
Nesopupa bisulcata
Pupisoma harpula
Pupisoma orcula
Pupisoma sp. A
Pupisoma sp. B
Acanthinula sp.
Rhachidina chiradzuluensis var. virginea
Conulinus rutshuruensis major
Cerastua trapezoidea lagariensis
Micrataeon koptawelilensis
Nothapalus sp.
Subulona clara
Oreohomorus iredalei
Pseudoglessula elegans
Pseudopeas cf. yalaensis
Curvella sp. A
Curvella cf. babaulti
Achatina stuhlmanni
Limicolaria cf. saturata
Punctum ugandanum
Punctum sp. A
Punctum sp. B
Trachycystis iredalei
Trachycystis ariel
Prositala butumbiana
Kaliella barrakporensis
Kaliella iredalei
carinate species
Guppya quadrisculpta
Afroconulus iredalei
Trochozonites cf. medjensis
Thapsia eucosmia
Thapsia microleuca
Thapsia spp.
Gymnarion aloysiisabaudiae
Chlamydarion oscitans
urocyclid slug
Halolimnohelix percivali
Halolimnohelix plana
Gonaxis elgonensis
Gulella woodhousei
Gulella osborni
Gulella impedita
Gulella ugandensis
Gulella lessensis
Gulella handeiensis
Gulella disseminata
Streptostele bacillum
Cecilioides sp.
Site totals
1952021
+
- +
+
+ -
+ +
+ +
+
+ =
+
5 +
+ +
+ +
+ +
+
+
+ +
+ 7
+ +
- +
+ +
+ +
+
+ +
+ +
+
+
+ +
+
+ +
+ +
+
+
+ -
+
22
++++++++
23 24 2526
+ + +
+
+
+
+ +
+
+ +
+ + +
+
+ + +
+ + +
+ +
+ +
+ +
+
+ + +
+
+
+ 7
+ + +
+ +
+ +
+ +
+
+ +
+
+ + + +
+ + + +
+ +
+
+
+ +
+ + + +
+ + + +
+ +
+
+ +
27 28 29 3031
+ + + +
+ + + +
+ + + + + + ++++ +
+
+++ ++ + + +
+
+
+++ ++
29 5 26 18 13 26 16 22 27 21 24 24 18
Species
totals
№
= BJ NN ND © ©
14
the Isiukhu River. Cecilioides is an open-
country genus, and the shells were probably
deposited onto the plots by the river. The
other species only recorded as dead shells
were Cerastua trapezoidea lagariensis, Gule-
lla handeiensis, Nothapalus sp., Trachycystis
MOLLUSCAN DIVERSITY IN À KENYAN RAIN FOREST 167
100
Mean
cumulative
percentage
of
50
total
species
in
5 10 15 20 29
Number of Plots
FIG. 2. Proportion of total indigenous forest fauna
(50 species) as a function of the number of plots
sampled.
ariel, Pupisoma sp. A and Rhachidina chirad-
zuluensis var. virginea; these have been in-
cluded in the analyses because they are
forest-dwelling species. Two species are
confined to the plantation plots and thus,
overall species number (S) in the indigenous
forest is 50. A plot of cumulative species
number against plot number (three random-
ized plot orderings) reaches an asymptote af-
ter 15 sites (Fig. 2). Because 27 indigenous
forest plots were sampled, it is clear that the
sampling detected the great majority of the
species present in the plots, unless some im-
portant micro-habitats containing specialist
species were overlooked.
The Fauna in Indigenous Forest
Possible groupings of plots have been in-
vestigated using Reciprocal Averaging Ordi-
nation (RAO) based on the presence and
absence data from all 31 plots. Species dis-
tribution and diversity levels have also been
examined in geographically restricted groups
of plots. RAO (Hill, 1973) is an ordination
method which arranges the plots along arti-
ficial axes according to their species comple-
ments. The ordination shows (Fig. 3) that the
faunas of most of the indigenous forest plots
are closely similar, except for a group of
seven plots which includes the Yala and Isi-
ukhu river plots (Plots 6, 7, 22, 23 and 24) and
Plots 8 and 10, which lie in Kisere and Mlaba
forests respectively. This smaller group
therefore appears to represent a forest-edge
fauna, which is either found in riverine forest
or in the smaller forest blocks; it is referred to
as the “riverine forest” group. Six species
are more frequent in the riverine forest group,
whereas four are more frequent in the larger
subset of indigenous forest plots (Table 2).
Plot 22 is particularly isolated on the ordina-
tion; it contains the only record for Notha-
palus sp. and is the only indigenous forest
plot to contain Limicolaria cf. saturata. The
species occurring in excess in the riverine
forest group are relatively large species,
whereas the four found in excess in the main
forest group are small litter-dwellers. The
plots containing disturbed or young indige-
nous forest fall within the main cluster of in-
digenous forest plots on the ordination, sug-
gesting that their fauna does not differ
substantially from that of the more mature
and less disturbed stands. However, one
species, Gulella impedita, is significantly less
frequent in the young forest than in the ma-
ture, undisturbed category (Fisher's Exact
Test, P < 0.05).
Species distribution throughout the study
area has also been investigated by plotting
site occupancy for each species on maps of
the forest, and by examining species fre-
quency in seven geographical groups of plots
(Table 3). These analyses essentially confirm
the findings of the ordination and show that
most species are widespread throughout the
forest. However, one species, Gulella ugan-
densis, is widespread in the survey area, oc-
curring from Mlaba to the Yala River, but is
apparently absent from the eight plots near
Isecheno; this pattern does not obviously re-
late to any of the environmental factors re-
corded and the reason for it is not known.
Patterns of Diversity in the
Indigenous Forest
Total species number varies amongst the
indigenous forest plots, but does not relate
obviously to geographical position or any of
the habitat or other environmental factors re-
corded. Mean species per plot (a diversity)
does not differ significantly amongst the
seven geographical groups (Table 3; Fé 18 ar
168
TATTERSFIELD
100 a a a
Аа LA
y 2
i= are € BA:
90 7 2°
a“ ae $ 24
Ze Riverine Forest Group ZAC
/ Oo -
ae o >
80 \ “cd
N $ Я
\ OREA
70
e
‘ 3
60
й O
Axis2 |e = é
$
Oo
50 o Bischofia
Pinus-Cupressus
©
40 e ево
30
20
10
KEY
PIE Indigenous forest plots
Plantation plots
10 20 30 40
50 60 70 80 90
Axis 1
FIG. 3. Reciprocal averaging ordination of the plots.
= 1.22, P = 0.34), between the riverine forest
group and the main indigenous forest groups
identified by the ordination (Table 3; F, 55 a+
= 0.16 P = 0.69) or between the undisturbed
mature indigenous forest, the young forest
and the highly disturbed indigenous stands
(Table 4; Fo 24 ar = 0.85, P = 0.44). There is
thus no evidence of significant variation in
alpha diversity levels throughout the survey
area or between the different age and distur-
bance categories of indigenous forest. Table
4 also gives values for Whittaker’s Index, /,
and the proportion of maximum variance
achieved for the sites per species statistic.
Neither of these measures suggests that
there are large differences between the plots
or that there is significant geographical re-
placement of taxa across the forest system.
Other Characteristics of the Fauna
The majority of the species recorded are
small litter-dwellers and were retrieved by the
sieving. Few species appeared to be re-
stricted to specific microhabitats. However,
Thapsia eucosmia was found almost exclu-
sively on living vegetation and on tree trunks,
Nesopupa bisulcata was most frequently re-
corded on the underside of dry, large, fallen
leaves on the forest floor, and Maizania elatior
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST
169
TABLE 2. Species recorded in significant excess in the riverine forest and other indigenous forest
groups identified by the ordination
Riverine forest
Number of Plots
Other indigenous
Species (Max = 7) (Max = 20) Probability*
More frequent in riverine forest group
Maizania elatior 7: 4 0.000
Cerastua trapezoidea lagariensis 5 2 0.005
Chlamydarion oscitans 9 4 0.023
Thapsia eucosmia 3 3 0.011
Conulinus rutshuruensis major 5 1 0.006
Gulella ugandensis 5 4 0.023
More frequent in other indigenous forest group
Nesopupa bisulcata 0 13 0.006
Curvella cf. babaulti 2 19 0.001
Curvella sp. A 2 16 0.023
Punctum ugandanum 1 15 0.009
*Probability based on Fisher's Exact Test. Null hypothesis that frequencies are equal in both groups.
TABLE 3. Mean number of species in geographical groups of plots and in two groups identified by
the ordination
Group Plots N Mean SE.
Geographical Groups
Mlaba 10, 11 2 27.00 3.00
Kisere 8, 28 2 24.00 3.00
Byungu' 12349 5 23.60 1.72
Isiukhu 6, 7 2 30.00 3.00
Isecheno 13, 14, 15, 16, 17, 18, 19, 21 8 2288 1.25
Yala 22, 23, 24, 27 4 21.00 3.34
Vihiga 29, 30 2 24.00 0.00
Ordination Groups
Riverine Forest 6, 7, 8, 10, 22, 23, 24 7 24.00 2.49
Other indigenous forest 1, 2, 3, 4, 5, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 20 23.20 0.83
27, 28, 29, 30
‘Excludes the young forest plots 5 and 12
was found on the surface of the forest floor
and appeared to have a highly aggregated
distribution. No snails were found amongst
epiphytes or on fallen tree branches, and no
evidence was detected that molluscs live in
the forest canopy.
About 73% of the species in the indigenous
fauna have shell sizes (maximum dimension)
of 10 mm or less and 52% are less than 5 mm.
About 17% have shell size exceeding 15 mm;
Achatina stuhlmanni is the largest species in
the fauna with a shell length in excess of 100
mm. In total, 3,723 specimens were collected
from the 27 indigenous plots, of which 1,504
(approximately 40%) were classified as living.
Guppya quadrisculpta constituted 10% and
11.25% ofthe living and overall totals respec-
tively and was the commonest species. Only
six species (12% of the total recorded fauna)
exceeded 5% of all the specimens (i.e., living
plus dead) recorded (Table 6) and these col-
lectively represented 46.23% of the overall
total. Twenty five species (50% of the fauna)
each contributed less than 1% to the total
number of specimens. The numbers of dead
and living specimens were very roughly equiv-
alent for most species, except for Subulona
clara and the aggregated Thapsia species,
of which many more dead shells than liv-
ing ones were found. During the fieldwork, it
was noted that substantial accumulations
of mostly dead Subulona clara shells were
170
TATTERSFIELD
TABLE 4. Estimates of mollusc diversity in indigenous forest and plantation
a
Indigenous Forest
Undisturbed, Disturbed, Young, recent
mature mature colonisation All indigenous Plantation
No. Plots 18 5 4 2, 4
Total species, S — — — 50 33
Species per site
Mean + SE, © 24.0 + 1.1 23.4 + 1.9 20.75 + 1.4 23.4 + 0.9 15.25 = 3:6
Range 13-33 19-30 18-24 13-33 5-22
Whittaker's Index, / — = — 2.1 2.2
Sites per species
Variance — — — 60.64 —
Maximum var. — — — 167.84 =
% achieved — — — 36.1% —
i gg;
TABLE 5. Species recorded in significant excess in the plantation and indigenous
forest categories
_Й—>-ррЁррЛЩЁЛЙ[ЩыырышышышышыыттштыяеьЁЙУУъчъъ»х‚х‚ххьььи—
Number of Plots
Indigenous
Plantation Forest
Species (Max = 4) (Max = 27) Probability*
More frequent in plantation
Limicolaria cf. saturata 2 1 0.037
More frequent in indigenous forest
Micractaeon koptawelilensis 0 24 0.001
Punctum ugandanum 0 16 0.043
Gulella osborni 1 25 0.008
Gulella disseminata 1 25 0.008
Gulella impedita 0 19 0.016
carinate species 0 26 0.000
*Probability based on Fisher's Exact Test. Null hypothesis that frequencies are equal in both plan-
tation and indigenous forest groups.
sometimes found in the leaf litter beneath rot-
ting and fallen logs.
Plantation Faunas
The small number of plantations sampled
inevitably means that conclusions are tenta-
tive. However, mean species per plot (Table
4) is significantly lower in the plantations than
in the indigenous forest (all 27 plots com-
bined) (F, 2о ах = 10.05, P = 0.004). Total
species number is exceptionally low in the
Pinus-Cupressus plantation. However, nei-
ther the Pinus-Cupressus nor the Maesopsis
plantations ordinate separately from the main
cluster of forest plots, although the two
Bischofia javanica plantations do (Fig. 3). Ta-
ble 4 gives Whittaker’s Index (/) for the
plantation plots; it does not differ substan-
tially from the value for the indigenous for-
est.
Six species are significantly more frequent
in the indigenous forest than the plantations
(Table 5), and all of these are small, litter-
dwellers; they are all also relatively abundant
in the indigenous forest (all being repre-
sented by at least 60 shells, Table 6). One
large species, Limicolaria cf. saturata, is sig-
nificantly more frequent in the plantations
than indigenous forest, and two others, the
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 171
TABLE 6. Number of specimens (living plus dead), proportion of total specimens collected and rank
order of each species in indigenous forest and plantation habitats
Indigenous forest Plantation
Rank Species Number % Number % Rank
1 Guppya quadrisculpta 419 1125 15 4.5 6
2 Thapsia Spp. 393 10.56 36 10.81 4
3 Gonaxis elgonensis 248 6.66 47 14.41 2
4 Pseudoglessula elegans 238 6.39 YA 251 14
5 Subulona clara 215 5.78 3 0.9 22
6 Pseudopeas cf. yalaensis 208 5:59 10 3 74
7 Kaliella barrakporensis 174 4.67 21 6.31 5
8 Kaliella iredalei 159 4.27 4 12 20
9 carinate species 153 4.11 0 — —
10 Maizania elatior 140 3.76 1 0.3 29
11 Curvella cf. babaulti 129 3.47 8 2.4 10
12 Gulella disseminata 100 2.69 5 125 18
13 Thapsia microleuca 96 2:58 3 0.9 22
14 Punctum ugandanum 76 2.04 0 — —
15 Gulella impedita 75 2.02 0 — —-
16 Micractaeon koptawelilensis 73 1.96 0 — —
7. Gulella ugandensis 71 1.91 49 16.72 1
18 Punctum sp. A 63 1.69 9 2.7 8
19 Gulella osborni 62 1.67 1 0.3 29
20 Gymnarion aloysiisabaudiae 59 1.59 7 2.1 14
21 Gulella woodhousei 58 1156 8 DT. 8
22 Curvella sp. A 57 1:53 8 2.4 10
23 Nesopupa bisulcata 52 1.4 0 — —
24 Thapsia eucosmia 50 1:35 0 — —
25 Streptostele bacillum 44 1.18 3 0.9 22
26 Trachycystis iredalei 33 0.89 1 0.3 29
27 Prositala butumbiana 22 0.59 2 0.6 26
28 Conulinus rutshuruensis major 21 0.56 4 1.2 20
29 Chlamydarion oscitans 20 0.54 8 2.4 10
29 Pupisoma harpula 20 0.54 2 0.6 26
31 Afroconulus iredalei 19 0.51 6 1.8 16
32 Punctum sp. B 17 0.46 0 — —
32 Gulella lessensis 17 0.46 0 — —
32 Truncatellina ninagongonis 17 0.46 0 = —
35 Pupisoma sp. A 15 0.43 0 -— —
36 Cerastua trapezoidea lagariensis 14 0.38 0 — =
36 Halolimnohelix plana 14 0.38 6 1.8 16
36 Pupisoma orcula 14 0.38 5 1.5 18
39 Elgonocyclus koptaweliensis 10 0.27 0 — —
39 Achatina stuhlmanni 10 0.27 3 0.9 22
39 Pupisoma sp. B 10 0.27 0 — —
42 Succinea sp. 9 0.24 0 = —
43 Oreohomorus iredalei 7. 0.19 1 0.3 29
44 Limicolaria cf. saturata 6 0.16 39 11.71 3
45 Trachycystis ariel 4 0:1 0 — —
45 Trochozonites cf. medjensis 4 0.11 2 0.6 26
47 Rhachidina chiradzuluensis 3 0.08 0 —
48 Acanthinula sp. 2 0.05 0 — —
49 Gulella handeiensis 1 0.03 0 — —
49 Nothapalus sp. 1 0.03 0 — —
urocyclid slug and Halolimnohelix percivali, tween the plantations and indigenous forest
are confined to the Bischofia plantation (al- (Table 6). In particular, Gulella ugandensis is
though in one plot only). The abundance of relatively much more abundant in the planta-
several species also differs substantially be- tions, whereas Guppya quadrisculpta and
172 TATTERSFIELD
Pseudoglessula elegans rank higher in the in-
digenous plots than in the plantations.
DISCUSSION
The Fauna of Kakamega Forest
The analysis of cumulative species number
suggests that the sampling has provided an
accurate picture of indigenous forest diver-
sity levels. However, many additional species
of terrestrial molluscs have been reported
previously from the Kakamega Forest area
(Germain, 1923; Pickford, 1995; Pain, 1957;
Verdcourt, 1983, 1988). A comprehensive ap-
praisal of these species would require critical
examination of all the available original ma-
terial and is beyond the scope of this study.
However, some species can be eliminated
from the list for the indigenous forest on the
grounds of conspecificity (e.g., see Pseudo-
peas yalaensis in Appendix |), and others
have almost certainly been misidentified. For
example, Germain (1923) lists Trachycystis
planulata Preston from “les bords de la
riviere Yala,” but because this species was
described from 9,000-10,000 on Mount
Kenya, it seems highly improbable that it oc-
curs at Kakamega. Verdcourt (1962) dis-
cusses another error made by Germain.
Some of these species may be associated
with non-forest habitats or ecotones and are
therefore not part of the indigenous forest
fauna. Furthermore, the need for major revi-
sions of some taxa, such as the genus Thap-
sia, means that it is not possible to make pre-
cise estimates of S for the forest. However,
by taking these factors into account as far as
possible, the total list for the forest might be
estimated at roughly 70-80 species. This
substantially exceeds the total species num-
ber (S) of 50 found in the indigenous forest
plots.
Local Patterns and Levels of Diversity
Solem (1984) has reviewed the evidence
on worldwide land snail diversity and has
shown that sympatric levels in most parts of
the world are low, with most sites typically
supporting less than ten species. However, a
few places are known to support much richer
faunas. The highest site diversity reported to
date is on the Manukau Peninsula, North Is-
land, New Zealand, where more than 60 spe-
cies have been found to exist microsympat-
rically in lowland patches of relict forest
(Solem et al., 1981). Solem (1984) also cites
the work of Fred Thompson and John Stani-
sic, who have recorded 25-30 species and
40 species from sites in the Greater Antilles
(Hispaniola and Jamaica) and from rain forest
in Queensland, New South Wales, respec-
tively. At least 50 species are present in some
Tanzanian coastal and upland forests (K. Em-
berton, pers. comm., and Tattersfield, un-
published). Alpha diversity can also be high in
rich temperate sites. Cameron (1986) reports
a median species number of 15 in coniferous
forest with mull humus soils in British Colum-
bia; Wáreborn (1969) assessed there to be a
mean snail species number of 16.58 per plot
in his richest Swedish woodlands; and
Waldén (1981) reported mean snail species
as 25.2 in five broad-leaved woodland site on
calcareous moraine in Sweden. Old wood-
lands in the Pennines (Cameron, 1978a) sup-
port a mean snail number of 28.25 species.
Values of S can also be high in optimum tem-
perate forests. Coney et al. (1982) reported
57 species from 37 forest sites in Tennessee;
Tattersfield (1990) found 31 species in En-
glish woodland sites on both limestone and
acidic geologies; and Branson 8 Batch (1970)
recorded a total of 45 species in Kentucky.
Based on these studies, it is therefore appar-
ent that the mean plot diversity of 24 species/
site and the overall total of 50 species from
the indigenous Kakamega stands are rela-
tively high in a worldwide context. Microhab-
itat specializations can help account for high
snail diversity in some faunas (Cameron,
1978b), but further work would be needed to
assess whether this is important in the Kaka-
mega fauna. However, the current study re-
vealed very few examples of possible micro-
habitat specializations.
Calculation of the Shannon-Weaver diver-
sity index (H) (Poole, 1974) and index of
evenness (J = H/Hmax), which take into
account the number of specimens contrib-
uted by each species as well as S, also indi-
cates that the Kakamega fauna is more di-
verse than almost all other woodland/forest
faunas that have been studied (Table 7); in-
deed, these indices show that it is on a par
with the richest known fauna described from
New Zealand (Solem et al., 1981).
Verdcourt, cited in Solem (1984), consid-
ered that “the wet forests of East Africa may
yield up to 20-25 species from a small area
although more frequently such a collection
yields only 10-15.” The only systematically
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 173
TABLE 7. Overall species number (S) and Shannon-Weaver diversity (H) and evenness
(J) indices for molluscan faunas from Kakamega Forest and other forest systems
Area/Forest type $
Kakamega Indigenous Forest
Live snails 43
All snails 50
New Zealand
Live snails 45
All snails 56
Tennessee, U.S.A. 57
British Columbia*
Mull litter sites 26
Intermediate litter sites 16
Mor litter sites 9
Finland (islands)
Deciduous woodland Site 9 21
Site 8 21
Site 6 19
Site 5 20
Sweden
Moist meadow woods 31
Drier mixed woods 9
Moist mixed woods 9
Shropshire, United Kingdom 12
*excludes slugs
collected information about mollusc diversity
patterns in African forests is from De Winter’s
(1995) series of 20 lowland rain forest plots
(from a total area of approx. 48 km?) in the
Ogoou, Maritime region of Gabon, West Af-
rica. Using similar methods to those adopted
here (snails extracted from approx. 4 | of lit-
ter), he reported mean species number per
litter sample as 3.4 (range 0-7, median 3),
with the number of specimens ranging from
0-20 (mean 7.9). lt was estimated that the
litter zone supported 28 (+ 2.2) species. A
similar number of species (about 25) in the
Kakamega fauna can be classified as small
(shell size < 5 mm) litter dwellers. Large spe-
cies were not sampled in plots so it is not
possible to compare directly alpha diversity
levels, but it is evident that many more spe-
cies and individuals were found in the Kaka-
mega litter samples than in Gabon; the aver-
age number of shells recovered from the
Kakamega indigenous forest plots was 137,
most of which were small litter-dwellers.
De Winter (1995) recorded a total of 32
species (including four freshwater species)
and estimated that the whole forest might
support 39 terrestrial species by taking into
account previous records; these values of S
are also lower than at Kakamega. Taking into
account the litter dwellers only, which form
82% and 51% of the recorded fauna in Kaka-
H J Source
3.23 0.86 This study
3.27 0.84
3.22 0.85 Solem et al. (1981)
3.26 0.81
2.93 0.73 Coney et al. (1982)
2.62 0.80 Cameron (1986)
27 0.78
1:72 0.78
2.60 0.85 Valovirta (1984)
2.35 0.77
2.26 0.77
2.25 0.75
2.31 0.67 Wareborn (1969)
1.41 0.64
1.23 0.56
1.75 0.64 Cameron (1982)
mega Forest and Gabon respectively, the ra-
tio S/ is substantially greater in Gabon than
at Kakamega. This indicates that there is
substantially more variation in the fauna
amongst the Gabonese plots than in Kaka-
mega Forest, but it is not possible to estab-
lish whether this is related to habitat variation
or to the geographical replacement of sister
species. Such a conclusion was also tenta-
tively made by De Winter (1995), who noted
that there were few species in common from
similar forest types over a distance of less
than 50 km, but that a fair number of addi-
tional species were found. Solem (1984) pre-
dicted that the median total linear range of all
land snails worldwide would be found to be
less than 100 km and probably less than 50
km, and Cameron (1992) has reported taxo-
nomic replacement effects over short dis-
tances in semi-arid habitats in Western Aus-
tralia. The commonest of De Winter’s (1995)
species was found in only half of the litter
samples, whereas 18 (36.7%) of the 49 indig-
enous forest species in Kakamega Forest
were found in more than 50% of the plots;
this also suggests that the fauna of the Ga-
bonese Forest is very much more heteroge-
neous than at Kakamega. In Kakamega
Forest, both / and the proportion of the max-
imum variance of the sites per species sta-
tistic achieved are broadly similar to the val-
174 TATTERSFIELD
ues reported by Cameron (1992) for faunas
from woodland in the English South Downs
(maximum separation 75 km) and from rock
habitats in the Pennines (separation 30 km).
They differ from British Columbian coastal
forests (maximum site separation of 300 km),
which have a more homogeneous fauna, and
from faunas from the Oscar and Napier
ranges of Western Australia (about 160 km
maximum separation), where both / (9.82)
and the proportion of variance achieved (8%)
revealed strong replacement effects in large
camaenid taxa (Cameron, 1992).
Origins of the Kakamega Forest Fauna
The African climate has been unstable dur-
ing the Pleistocene, and this had a strong
influence on forest cover. Many parts of equa-
torial Africa were dry and cool during the last
ice age, and Lake Victoria was almost non-
existent at around 14000 BP, during the Last
Glacial Maximum in Africa (Kendall, 1969; Liv-
ingstone, 1980). With the absence of mois-
ture-laden convection currents from the Lake
Victoria waterbody, the Kakamega area
would not have supported forest cover. There
is evidence (reviewed in Hamilton, 1982) that
forest in equatorial Africa became confined to
a relatively small number of discrete areas
during times of extended glaciation and that
the current distribution patterns of many
groups of forest species can be accounted for
by subsequent expansion from these refugia
(Hamilton, 1982; Kingdon, 1990). Former for-
est refugia are now often rich in endemic spe-
cies. The Gabonese forest studied by De
Winter (1995) falls within or close to such a
refugium at the Gabon-Cameroon border
(Kingdon, 1990), whereas Kakamega Forest
does not. The mollusc fauna of Kakamega
Forest must have recolonised after the cli-
matic amelioration and redevelopment of for-
est. Palynological evidence (Hamilton, 1972;
Kendall, 1968; Livingstone, 1967) shows that
at 12000-10000 years BP, forest spread from
a refugium in eastern Zaire (possibly extend-
ing into west Uganda) across what is now
Uganda and the Kakamega area of west
Kenya. Furthermore, studies on the avifauna
(Zimmerman, 1972) and tree flora (Hamilton,
1982) of Kakamega Forest have shown that
they are impoverished versions of the Central
African biotas, which also strongly suggests
that recolonisation was from west or central
Africa. The mollusc fauna of Kakamega Forest
would suggest a similar route of recolonisa-
tion because Pain (1957) has shown that
Achatina stuhlmanni is commonest in Zaire
west of the Upper Ituri River and the genera
Prositala, Pseudoglessula (Ischnoglessula),
Oreohomorus, Nothapalus, Conulinus, Tro-
chozonites (Zonitotrochus), and Gulella (sect.
Silvigulella) all have west and central African
affinities (Verdcourt, 1972).
Regional Patterns and Levels of Diversity
The mollusc fauna of East Africa (Kenya,
Uganda and Tanzania) contains about 1,015
terrestrial species of which about 844 (83%)
are forest dwellers (Verdcourt, 1972). Based
on the current survey results plus other
records it can therefore be assessed that the
fauna of Kakamega Forest supports about
5.8-9.5% of the potential forest fauna of the
region. This low proportion indicates that
there must be substantial geographical re-
placement of taxa throughout the region. As
discussed above, this situation is probably
not unusual worldwide; Solem (1984) sum-
marised the available evidence which sug-
gests that allopatric diversity is exceptionally
high amongst land snails. The regional levels
and patterns of terrestrial snail diversity are
very different in temperate northwest Europe,
where the fauna is relatively homogeneous
over large geographical areas (Cameron,
1995). Kerney & Cameron (1979) cover a land
area about 38% larger than East Africa, but it
supports only 279 terrestrial molluscs (Ker-
ney 8 Cameron, 1979), of which about 152 or
54.5% are forest or woodland species. Rich
sites in Britain may support a large propor-
tion of the national gastropod fauna; for ex-
ample, Whitcombe Wood on Jurassic lime-
stone in Gloucestershire (Boycott, 1934)
supports 37 gastropods (28 snails and nine
slugs), which represent approximately 33%
of the total British land gastropod fauna (or
32% of the snail fauna).
Bernard Verdcourt (in Rogers & Home-
wood, 1982) lists 111 terrestrial gastropod
species and subspecies known from the
Usambara Mountains in northern Tanzania.
These mountains support lowland and inter-
mediate rain forest communities and have
rainfall (1919 mm/year at Amani (911 m asl))
and temperature (21.7-28.3°C mean maxi-
mum at Amani) regimes broadly rather similar
to Kakamega Forest. However, the list in
Rogers 4 Homewood (1982) has only seven
species in common with the total reported
fauna (i.e., all records) from Kakamega For-
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 175
est. Unlike Kakamega Forest, the Usambaras
are thought to have have supported forest
cover for millions of years, probably since be-
fore the Miocene, and contain very high num-
bers of endemic species (Rogers 4 Home-
wood, 1982). The richer overall fauna in the
Usambaras may be related to this long period
of forest stability, plus its greater extent and
more diverse physical geography and habi-
tats. However, at individual sites in the East
Usambara, values of both S and « are
broadly similar to those found in Kakamega
Forest (Tattersfield, unpublished).
Plantation Faunas
Impoverished snail faunas and low mollus-
can diversity levels have been reported from
plantations elsewhere (Cameron, 1978a). The
very small number of plantations examined
restricts firm conclusions, but there is evi-
dence that both the Maesopsis and Bischofia
plantations also have impoverished faunas,
and that the latter also appears to be com-
positionally different from the indigenous for-
est. Differences in the diversity and species
composition in several groups of soil arthro-
pod have been reported between primary
forest and Maesopsis eminii plantation in the
East Usambaras in Tanzania (Mahunka,
1989). Maesopsis eminii, which is not an in-
digenous species in northeast Tanzania and
where it is considered to be an ecological
weed, has also been shown to alter radically
the characteristics of the litter and topsoil
(Hamilton, 1989) and to be associated with a
loss of the organic soil horizons (Macfadyen,
1989; Binggeli & Hamilton, 1993). Whether
Maesopsis itself or other factors, such as
canopy loss, drainage or disturbance, is re-
sponsible for organic horizon loss is not clear
(Macfadyen, 1989), although such changes
might be expected to have a large effect on
the small, litter-dwelling snails, in line with
that reported here. The six species (Table 5)
that are less frequent in the plantations may
thus, tentatively, be regarded as indicators of
indigenous forest, in the same way that some
species can be used to assist in the differen-
tiation of ancient and secondary woodland in
Britain (Kerney 8 Stubbs, 1980). Lovejoy et
al. (1986) have demonstrated that substantial
changes occur in microclimate at the edges
of recently cleared rainforest. It is interesting
in this context that the species that are sig-
nificantly less frequent in both the riverine
(forest edge) indigenous plots and in the
plantations are all small, litter dwellers that
might be expected to be more suceptible to
such changes.
Conservation Implications
The mollusc fauna of Kakamega Forest
does not contain the high numbers of en-
demic species found in some other forest
systems in East Africa (for example the Tan-
zanian Usambara ranges (Rogers 4 Home-
wood, 1982)). However, in common with the
bird and butterfly faunas and the flora, its ma-
lacofauna does support central and west Af-
rican elements, which are scarce or absent
from most of Kenya and Tanzania. The mol-
luscs of Kakamega Forest are therefore of
some biogeographical interest, and they sup-
plement this previously acknowledged con-
servation importance of the Kakamega For-
est system.
Further information is required about local
diversity patterns in other African forests, but
if faunas from other forest systems are rela-
tively uniform like in Kakamega Forest, then
this, and the high level of allopatric diversity
throughout East African forests, have several
potential conservation implications. Notwith-
standing the probable importance of edge ef-
fects in forest fragments, the majority of mol-
lusc species found during the study could
probably be conserved in a relatively small
area of forest. Of course, there are many im-
portant reasons why the size of protected ar-
eas should be maximised, but based on
these conclusions and from a solely mollus-
can perspective, it is apparent that the pro-
tection of a large number of widely distrib-
uted, small forest blocks might be more
effective at conserving regional molluscan
biodiversity than would the retention of a
smaller number of large forest areas of equal
extent. The degree to which regional mollusc
biodiversity could be maintained by conserv-
ing the endemic-rich forest systems needs
further survey and analysis.
ACKNOWLEDGEMENTS
This project could not have been under-
taken without the help given by many people,
all of whom | would like to thank. In particular,
| would like to thank Dr. Bernard Verdcourt
(Kew), who helped with the identifications
and shared his immense knowledge about
East African molluscs. Drs. Mary Seddon and
176 TATTERSFIELD
Graham Oliver of the National Museum of
Wales, and Dr. Peter Mordan, Fred Naggs
and Kathie Way of The Natural History Mu-
seum (Malacology Section) kindly made their
extensive collections available and also of-
fered advice and assistance. Professor Rob-
ert Cameron (Sheffield) and Dr. Laurence
Cook (Manchester) provided advice and sup-
port, and Dr. A. C. van Bruggen (Leiden) as-
sisted with identification. Computer and
other facilities were made available by Penny
Anderson Associates. Gene Hammond
helped with the drawings. Mollusc speci-
mens were kindly loaned by V. Heros (MNHN,
Paris), W. K. Emerson (AMNH, New York),
Tanya Kausch (MCZH, Harvard) and Dr. R.
Kilias (Museum für Naturkunde der Humboldt
Universität, Berlin).
| would like to thank the Office of the Pres-
ident of Kenya for permission to undertaken
the research. Dr. Richard Bagine and Mu-
sombi Kibberenge (National Museums of
Kenya) kindly provided advice and help when
organising the fieldwork. The Kenya Wildlife
Service gave support in the field. Dr. Glyn
Davies and other members of the KIFCON
team provided information about Kakamega
and other Kenyan forests. | am also very
grateful to my local assistants, Nixon Sagita,
Solomon Astweje and Caleb, who both
helped with sampling and were invaluable
guides in the forest.
The work was kindly supported by grants
from the British Ecological Society/Coal-
bourn Trust, the People's Trust for Endan-
gered Species and the Percy Sladen Fund of
the Linnean Society. Ross Lab of Maccles-
field and J. G. Stanniar 4 Co., Manchester
generously supplied equipment.
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Revised Ms. accepted 1 March 1996
APPENDIX I. SPECIES NOMENCLATURE
AND NOTES ON IDENTIFICATION
Museums are referred to in this appendix as
follows:
BMNH — Natural History Museum, London,
United Kingdom
NMW — National Museum of Wales, Cardiff,
United Kingdom
AMNH — American Museum of Natural His-
tory, New York, U.S.A.
MCZH — Museum of Comparative Zoology,
Harvard, Cambridge, Mass., U.S.A.
MNHN — Muséum Nationales d'Histoire Na-
turelles, Paris, France
Systematic List
Elgonocyclus koptaweliensis
Verdcourt (1982a, 1991a).
Maizania elatior (von Martens) — Verdcourt
(1964).
(Germain) —
“Succinea” sp. — A revision of African Suc-
cineidae is required before the genera
present can be elucidated or the species
named consistently. The genus Suc-
cinea Drap. probably does not occur in
Africa (Verdcourt, 1972).
Truncatellina ninagongonis (Pilsbry) — The
Kakamega material matches the holo-
type (MCZH 77268) collected from Mt.
Ninagongo, 9000 ft. (north of Lake Kivu),
Zaire (Tattersfield, 1995).
Nesopupa bisulcata (Jickeli) — Adam (1954)
and Bruggen 4 Verdcourt (1993).
Pupisoma (Salpingoma) harpula (Reinhardt)
(= Pupisoma japonicum Pilsbry) — Iden-
tification by Dr. A. C. van Bruggen (Lei-
den). Also Adam (1957). This widespread
species is not listed in Verdcourt (1983).
Pupisoma orcula (Benson) — Adam (1957)
and material in NMW from Bekar, India.
Pupisoma sp. À — Shell almost globular (с.
2.2 x 2 mm), brownish olive-green, with
fine radial sculpture.
Pupisoma sp. B — Probably a Pupisoma but
matches none of the species in Adam
(1954, 1957). However, the species (ap-
prox. 2 x 1.4 mm) has a moderately large
umbilicus and traces of lamellae on the
periphery, which make it rather endo-
dontoid in appearance.
Acanthinula sp. — The Kakamega material
has only a few small spines. It lacks the
spiral striation on the first whorl of both
Preston's expatriata Preston (holotype,
BMNH 1937.12.30.2085) and Pilsbry’s
azorica; it may be underscribed.
Rhachidina chiradzuluensis var. virginea
(Preston) — Matches Preston's cotypes
(BMNH) from Mount Kenya.
Conulinus rutshuruensis major Verdcourt —
Matches paratype (BMNH 196731,
Nandi Forest, Kenya).
Cerastua trapezoidea lagariensis (E. A. Smith)
Micractaeon koptawelilensis (Germain) (=
kakamegaensis Verdcourt) — Verdcourt
(1990).
Nothapalus sp. — Shell yellow, 19.5 x 6.4
mm. lt seems unwise to name this spe-
cies on the basis of the single specimen.
The shell of Preston’s iredalei is narrower
and his suturalis is larger. However, it is
not disimilar in shape to either of these
species. lt has not been possible to
compare the shell with material of N. ba-
baulti (Germain) or N. paucispirus (von
Martens).
Subulona clara Pilsbry — The Kakamega
MOLLUSCAN DIVERSITY IN A KENYAN RAIN FOREST 1149
species matches material of clara in
BMNH.
Oreohomorus iredalei Preston — The size
and shape of the Kakamega specimens
match material from the Belgian Congo
and Mount Elgon in NMW, although
shells of the former have more white co-
louration. This species is conspecific
with O. nitidus (von Martens) (Verdcourt,
1983). Examination of a syntype of O.
albini Germain (MNHN), which was de-
scribed from Kakamega Forest, indi-
cates that it is close or identical to the
material collected in 1993, although it
has lost its periostracum. The strong
crenulations and spiral sculpture on the
early whorls described by Germain
(1923) and shown on the illustration are
not visible even though the syntype ex-
amined is clearly the illustrated shell.
Pseudoglessula (Ischnoglessula) elegans
(von Martens) — Kakamega material
matches syntypes of elegans (BMNH),
although all the types are in very poor
condition with bleached or missing peri-
ostracum. The material from Kakamega
also appears identical to the illustration
of P. subfuscidula Pilsbry, 1919, which is
probably conspecific (Verdcourt, 1983).
Verdcourt (1983) lists P. mutandana
Connolly from Kakamega Forest but this
species (syntype in BMNH) is larger and
has much finer ribbing on the first four
whorls than elegans; it was not found
during the survey.
Pseudopeas cf. yalaensis Germain — The
Kakamega specimens match syntype
material of yalaensis (MNHN); the types
have very faint and barely perceptible
spiral sculpture on the first whorl. Opeas
euschemon Connolly may be conspe-
cific (Verdcourt, 1983); however, the six
shells of this species (NMW, Melvill and
Tomlin coll., Mt. Mikeno) are larger than
the types of yalaensis, and there is no
trace of spiral sculpture. Their general
shape is however similar and further ma-
terial would be needed to confirm
whether they are the same species.
Curvella sp. À — shell broadly conic, thin,
transluscent white, approx. 7 x 3.5 mm.
Apical whorls smooth, remainder with ir-
regular, arcuate growth lines. Outer lip
curved in profile, arching forward in the
centre. Columella curving smoothly into
the basal margin of the shell mouth, not
truncate.
Curvella cf. babaulti Germain — The Kaka-
mega specimens appear identical to the
syntype (MNHN). The Kakamega mate-
rial also matches Pseudopeas ke-
kumeganum Connolly (syntype in
BMNH), which Verdcourt (1983) sug-
gests may be conspecific. The Kaka-
mega shells have faint spiral micro-
sculpture indicating that the species
belongs in Pseudopeas.
Achatina stuhlmanni von Martens — Pain
(1957).
Limicolaria cf. saturata Smith — The Kaka-
mega material has a similar shape to the
saturata holotype although the shells are
smaller. Material of saturata in NMW also
generally has larger shells than the
Kakamega specimens and further inves-
tigations are desirable to confirm identi-
fication.
Punctum ugandanum (E. A. Smith) — Verd-
court (1988).
Punctum sp. A— The shells (approx. 1.2 x
0.8 mm) have rather regular ribbing and
a characteristic spiral micro-sculpture
suggesting that the species is in Punc-
tum. Possibly close to hottentotum
(Melvill & Ponsonby) but spire more ele-
vated.
Punctum sp. В — Shell (с. 1.6 x 0.9 mm) with
lamellae and possibly in Trachycystis.
Smaller, less elevated spire and without
the very broad lamellae of E. A. Smith’s
lamellifera.
Trachycystis iredalei
(1991, ©).
Trachycystis ariel (Preston) — Agrees with
paratype in BMNH. Also illustration in
Bruggen (1969).
Prositala butumbiana (von Martens) — Verd-
court (1991b, c).
Kaliella barrakporensis (Pfeiffer)
Kaliella iredalei Preston
Carinate species (undescribed) — This dis-
tinctive but undescribed, minute species
is distributed widely across Africa
(Malawi, Zaire, Angola, Ghana and vari-
ous other west African countries) (pers.
comm., A. C. van Bruggen). The shell is
discoid and has six, spiral lamellae. The
Kakamega material appears to be poly-
morphic for shell colour with both white/
transparent and red-brown shells.
Guppya quadrisculpta (Connolly)
Afroconulus iredalei (Preston) — Micro-
scopic and larger-scale shell sculpture
and shape agree very well with cotype of
Preston — Verdcourt
180 TATTERSFIELD
iredalei (BMNH, Mt. Kenangop, Aber-
dares, Kenya). However, the Kakamega
material also does not differ significantly
from urguessensis (Connolly), which may
therefore be conspecific. The type of A.
diaphanus (Connolly) could not be found
in BMNH.
Trochozonites (Zonitotrochus) cf. medjensis
Pilsbry — The angle of the shell apex is
smaller and the ribbing stronger in the
Kakamega material than in the holotype
(AMNH, Medje, Belgian Congo). How-
ever, overall shape and size agree well.
The holotype shell of expatriata Preston
(BMNH, Mt. Mikeno, Belgian Congo) has
a much flatter base than both medjensis,
and the Kakamega species and is clearly
different.
Thapsia eucosmia Pilsbry — Agrees well with
the holotype (AMNH, Medge, Belgian
Congo). This Thapsia has a “'nipple-like”
apex to its shell and shouldered whorls,
which appear effectively to separate it
from the other species collected during
the study.
Thapsia microleuca Verdcourt — Verdcourt
(1982b).
Thapsia spp. — Preston figured many spe-
cies in this genus, which appear to be
barely separable even with type material
side by side for comparison. In the ab-
sence of a full revision of the genus, it
seems unwise to assign names to the
Kakamega material. In addition to eu-
cosmia and microleuca, the following
species have been recorded from Kaka-
mega forest previously:
Thapsia elgonensis (Preston)
Thapsia cinnamomeozonata Pilsbry
Thapsia densesculpta (Preston)
Thapsia karamwegasensis Germain
Thapsia yalaensis Germain
Thapsia gerstenbrandti (Preston) ?= elgonen-
sis (Prest.)
Thapsia mime (Preston)
There appears to be at least two unidentified
species in the Kakamega material. One
has strong spiral striae and tight whorls
without any evidence of a shell band.
The other has faint spiral microsculpture
and a brown shell with a faint band; this
species is close to mime (Preston).
Gymnarion aloysiisabaudiae (Pollonera)
Chlamydarion oscitans (Preston)
Urocyclid slug
Halolimnohelix percivali (Preston) — Con-
firmed by dissection by B. Verdcourt.
Both holotype and paratypes (BMNH)
are juvenile and lack a reflected peris-
tome, but the shells otherwise agree with
the Kakamega material.
Halolimnohelix plana Connolly — Agrees
with holotype in BMNH; also Verdcourt
(1981).
Gonaxis elgonensis (Preston) — material
agrees well with paratypes in BMNH.
Gulella woodhousei (Preston), = babaulti
Germain, ?= perturbata Preston.
Gulella osborni Pilsbry — Illustrated by Pils-
bry (1919).
Gulella impedita Connolly — agrees perfectly
with holotype in BMNH.
Gulella lessensis Pilsbry — Illustrated by Pils-
bry (1919).
Gulella handeiensis Verdcourt
Gulella disseminata (Preston) — The Kaka-
mega material agrees well with the holo-
type of var. kekumegaensis Connolly
(BMNH).
Gulella ugandensis (E. A. Smith) — See Verd-
court (1970).
Streptostele bacillum Pilsbry — The Kaka-
mega material has spiral microsculture
on the apical whorls. It matches perfectly
with the holotype (AMNH, Bequaert
Coll.) collected from lturi Forest, Penge,
Zaire (Pilsbry, 1919).
MALACOLOGIA, 1996, 38(1-2): 181-202
MOLECULAR GENETIC IDENTIFICATION TOOLS FOR THE UNIONIDS OF
FRENCH CREEK, PENNSYLVANIA
Laura В. White,’ Bruce A. McPheron,? & Jay В. Stauffer, Jr.'
ABSTRACT
A molecular genetic key to the unionids of French Creek, Pennsylvania, an Allegheny River
tributary, is presented here. The key is an integral part of a new approach to identifying unionid
glochidia larvae attached to host fishes in the drainage. Working with tissue from adult union-
ids, we used the polymerase chain reaction (PCR) followed by restriction enzyme digests to find
species-specific genetic “fingerprints” for the 25 species in the drainage. We have demon-
strated the utility of the key by using it to identify 70 glochidia attached to fishes collected in the
French Creek drainage.
Key words: Unionoidea, glochidial identification, PCR, RFLP analysis, ITS regions.
INTRODUCTION
North America’s freshwater mussels (Bi-
valvia: Unionoidea) are declining precipi-
tously in richness and abundance (e.g., Den-
nis, 1987; Anderson et al., 1991; Nalepa et
al., 1991; Williams et al., 1992, 1993). Sizable
gaps in knowledge of unionid reproductive
requirements hamper current preservation
efforts. Information on the identities of the
host fishes upon which unionid glochidia lar-
vae are obligate parasites is especially in-
adequate. Traditional methods of gathering
such data have a variety of drawbacks.
To date, lists of unionid host fishes have
been derived primarily in two ways. The first,
which has its roots in artificial propagation
efforts (e.g., Lefevre 8 Curtis, 1910, 1912;
Coker et al., 1921), involves inoculating pu-
tative hosts with glochidia taken from gravid
females of the unionid species of interest.
Fishes in aquaria that ultimately contain meta-
morphosed juveniles are considered suitable
hosts (e.g., Zale 8 Neves, 1982; Waller & Hol-
land-Bartels, 1988). Unsuitable hosts launch
immune responses that thwart glochidial en-
cystment, preventing further development
and causing glochidia to be shed (Arey,
1923a, 1932).
As the completion of metamorphosis re-
quires a week to several months of attach-
ment (Zale & Neves, 1982), this approach is
often time-consuming. It is also ill-suited to
systems with large numbers of potential host
fishes. Moreover, drawing inferences from in-
oculation studies can be complicated by the
fact that “suitable” host fishes can appar-
ently acquire immunity to glochidia with re-
peated exposure, the duration and species
specificity of which are poorly established
(Reuling, 1919; Arey, 1923b; Fuller, 1974). To
obtain unambiguous results, it is often nec-
essary to collect putative hosts from unionid-
free streams or to inoculate naïve fishes bred
and raised in the laboratory. Finally, while
artificial inoculation methods are appropriate
if laboratory propagation of unionids is the
only goal, the results of such studies might
be inapplicable to organisms in their natural
environments. Such studies disregard micro-
habitat preferences and specialized mor-
phologies and behaviors (e.g., the waving of
fish-like mantle flaps by gravid female Lamp-
silis species; Ortmann, 1911; Kraemer, 1970)
that might modulate unionid-fish interactions
in situ.
To circumvent these problems, several in-
vestigators (e.g., Wiles, 1975; Stern & Felder,
1978) have attempted morphology-based
identification of glochidia attached to fishes.
Such determinations have thus far entailed
identifying the glochidia using dissecting mi-
croscopes or compound light microscopes.
There are drawbacks to this approach as
well. Glochidia are less than 1 mm in diameter.
Encystment makes them difficult to observe
and might influence their shapes in unpredict-
able ways (Wiles, 1975). Closely related spe-
'School of Forest Resources and Intercollege Graduate Degree Program in Ecology, 2C Ferguson Bldg., The Pennsylvania
State University, University Park, Pennsylvania 16802, U.S.A.
Department of Entomology and Institute of Molecular Evolutionary Genetics, 535 ASI Bldg., The Pennsylvania State
University, University Park, Pennsylvania 16802, U.S.A.
182 WHITE, MCPHERON 8 STAUFFER
cies, such as Villosa nebulosa (Conrad) and
Villosa vanuxemensis vanuxemensis [vanux-
emi] (|. Lea) (Zale 4 Neves, 1982), are difficult
to distinguish from each other and are easily
misidentified. Hoggarth (1992) reported that
glochidia photographed by Wiles (1975) and
identified by the author as Pyganodon [An-
odonta] cataracta (Say) were actually Alasmi-
donta undulata (Say). Clarke (1981, 1985),
Rand & Wiles (1982), and Hoggarth (1988)
demonstrated that scanning electron micros-
copy can be used to distinguish among
glochidia taken from gravid females. Whether
their techniques can be adapted for species-
level identification of glochidia from host
fishes remains to be investigated, however.
The objective of the research described
herein was to develop a new method for
identifying glochidia attached to fishes, a
method that exploits genetic differences
among unionid species. The method utilizes
restriction fragment length polymorphism
(RFLP) analysis of polymerase chain reaction
(PCR) products. In combination, PCR and
RFLP analysis are useful for performing sen-
sitive analyses of minute quantities of DNA
(e.g., Whitmore et al., 1992; Simon et al.,
1993), such as those present in single
glochidia. In short, a diagnostic suite of re-
striction sites (or “genetic fingerprint”) is
sought for each unionid species in the drain-
age of interest. Glochidia on host fishes are
then identified on the basis of the “finger-
prints” they possess.
MATERIALS AND METHODS
Study Site
The aquatic system for which the glochidial
identification method was developed is the
French Creek drainage, in southwestern New
York and northwestern Pennsylvania (Fig. 1).
French Creek is a fourth-order tributary to the
upper Allegheny River. It drains approxi-
mately 3,000 km”. Twenty-five unionid spe-
cies (C. Bier, pers. comm.) and 53 fish spe-
cies (J. Stauffer, unpubl. data) have been
collected from the French Creek drainage re-
cently, making its fish and molluscan faunas
the richest in Pennsylvania. Two of the drain-
age's unionid species, Epioblasma torulosa
rangiana (l. Lea) and Pleurobema clava (La-
marck), are federally endangered and have
no known hosts. Two additional species are
considered globally threatened and seven
are of special concern (Williams et al., 1993);
of these nine, five have no known hosts.
LeBoeuf Creek is thought to harbor higher
densities of P. clava than any other part of the
drainage (A. Bogan, pers. comm.). To assess
the utility of the identification technique,
fishes were collected from LeBoeuf Creek at
Moore Road bridge, just east of Route 19, 3
km south of LeBoeuf Gardens, Pennsylvania
(Fig. 1). Full descriptions of the site and col-
lection procedures are given by White (1994).
Specimen Collection and Preservation
Adult unionids. Adult unionids were col-
lected throughout the French Creek drainage
(Fig. 1, Table 1) in 1991, 1992, and 1993.
Numbers of unionids collected ranged from
one to 23 per species, with a median of six.
Adult Lasmigona costata (Rafinesque), Am-
blema plicata (Say), and Lampsilis siliquoidea
(Barnes) specimens were also collected from
West Virginia (Dunkard Creek) and Ohio
(lower Muskingum River, Little Muskingum
River, and Big Darby Creek), so that their ge-
netic “fingerprints” could be compared with
those of French Creek specimens to evaluate
the key's applicability to other drainages.
Adult unionids were collected using masks
and snorkels or Plexiglas-bottomed buck-
ets. Nonendangered species were trans-
ported to the laboratory either alive (wrapped
in cheesecloth in chlorine-free ice water) or
frozen on dry ice. In the laboratory, live union-
ids were either killed and frozen at —80°C, or
maintained in aquaria in which currents were
established. Two small (5- to 100-mg) pieces
of foot tissue were excised from each indi-
vidual in the laboratory using a sterile scalpel
blade or scissors. Both samples were frozen
at —80°C, one for nucleic acid extraction and
the other for voucher material. The remaining
tissue was preserved with the valves in 70%
ethanol, also as voucher material. To facilitate
future molecular genetic examination, the lat-
ter tissue was not fixed in formalin. All voucher
material was deposited into the mollusc col-
lection of the Academy of Natural Sciences in
Philadelphia upon completion of the research
(Dry Catalog # 398499-398500; Alcohol Cat-
alog + A18354-A18438; Frozen Catalog +
F100-F118).
For endangered unionids, a single tissue
sample was obtained from each specimen at
streamside by relaxing its adductor muscles
in soda water and clipping off a 5- to 50-mg
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 183
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WHITE, MCPHERON & STAUFFER
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MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 185
piece of foot using a sterile scalpel blade or
scissors (Pennsylvania Fish and Boat Com-
mission permit number 142 (Type |); proce-
dure reviewed prior to permitting by the
United States Fish and Wildlife Service). Tis-
sue samples were frozen immediately on dry
ice for transportation to the laboratory, where
they were kept at —80°C pending nucleic
acid extraction. After a 10- to 15-min recov-
ery period in a bucket of streamwater, the
specimens were photographed and returned
to natural positions in the substrate as close
to their original locations as possible.
Fishes. Fishes were collected throughout
French Creek by kick-seining and were trans-
ported to the laboratory on dry ice. In the
laboratory, a 5- to 100-mg piece of muscle
was excised from the body wall of each and
was frozen at —80°C prior to nucleic acid
extraction. The remainder of each specimen
was also frozen at —80°C as voucher mate-
rial.
Glochidia. Glochidia of Known identity were
obtained from marsupia of gravid nonendan-
gered female unionids collected and frozen
as described above. Glochidia of unknown
identity were obtained from fishes collected
throughout French Creek by kick-seining.
The fishes were transported to the laboratory
alive, maintained in an aquarium for one
week, then killed and frozen at —80°C; un-
encysted glochidia were presumed to have
been shed during the holding period. En-
cysted glochidia were removed as described
below.
Laboratory Techniques
Nucleic acid extraction. For adult unionids,
unattached glochidia, and fishes, a standard
phenol-chloroform extraction protocol (after
Kocher et al., 1989) was used to isolate total
nucleic acids. Each tissue sample was
minced over ice using a sterile scalpel blade,
then transferred to a 1.5-ml microfuge tube
and homogenized in 500-800 ul of extraction
buffer (100 mM Tris-HCl, pH 8.0; 10 mM
EDTA; 125 mM NaCl; 0.1% SDS; 50 mM
DTT; 5 ug/ul proteinase K) using a flame-
sealed 1000-ul pipette tip; different scalpel
blades and pipette tips were used for each
sample, to prevent cross-contamination. Ho-
mogenized samples were incubated 2-24 hrs
at 37°C, then extracted sequentially with
equal volumes of Tris-buffered phenol, 50%
phenol-50% chloroform, and chloroform (=
24 chloroform: 1 isoamyl alcohol, v:v; Sam-
brook et al., 1989). Samples were centrifuged
4-5 min at 16,000 x д during each extraction
to separate the phases. After the final extrac-
tion, 0.05 volume of 5 M ammonium acetate
and two volumes of cold absolute ethanol
were added to each sample. Samples were
placed at —80°C for 15-30 min, then spun
15-45 min at 16,000 x g at 4°C. Supernatants
were decanted and pellets were dried in a
Savant SpeedVac Concentrator. Pellets were
resuspended in 10-25 ul of sterile distilled
water, depending on their size, and stored at
—20°C. Even when no pellet was visible in a
tube, 10 ul of sterile distilled water was
added and the sample was stored at — 20°C.
Extractions were assayed on 0.8%-agarose
minigels stained with ethidium bromide and
were diluted 0-1000х depending upon esti-
mated DNA concentration.
For glochidia attached to fishes, an extrac-
tion protocol similar to that described by
Martin et al. (1992) for fish oocytes was used.
Each glochidium was removed from its host
over ice using sterile forceps and a dissecting
light microscope, then transferred with a
200-ul pipette tip to a 1.5-ml microfuge tube
containing 30 ul of buffer (50 mM KCI; 10 mM
Tris-HCl, pH 8.3; 1 pg/ml proteinase К; 1
ug/ml bovine serum albumin). Nonidet P-40
was added to a final concentration of 1%.
Solutions were heated to 95°C for 5 min т a
thermal cycler, diluted to a final volume of 50
ul with sterile distilled water, and stored at 4°
or —20°C. Extractions were not assayed
prior to amplification, as they contained too
little DNA to be visualized with ethidium-bro-
mide staining (data not shown).
Amplification. Reaction volumes of 50 or 100
ul were used. Reaction mixtures consisted of
0.5-2.0 ul of diluted template DNA; 1 uM of
each primer (0.2 uM of each RAPD primer);
0.1 mM each of dATP, dCTP, dGTP, and
dTTP; 2.0-2.5 units of Perkin-Elmer Cetus
Taq polymerase; and manufacturer-supplied
buffer at 1х final concentration (10 mM Tris-
НС, pH 8.3; 50 mM КС; 15 mM MgCl,;
0.01% (w:v) gelatin). For glochidia from host
fishes, 1-10 ul of undiluted template was
used.
Primer sequences were as follows. ITS-1
of nuclear rDNA: 5’-TAACAAGGTTTCCG-
TAGGTG-3’ (18S region) and 5’-AGCTRGC-
TGCGTTCTTCATCGA-3’ (5.85 region); ITS-1
through ITS-2: 5’-TCCGTAGGTGAACCTGC-
186 WHITE, MCPHERON & STAUFFER
GG-3’ (ITS1 of Lee & Taylor, 1992; 18S region)
and 5’-TCCTCCGCTTATTGATATGC-3’ (ITS4
of Lee & Taylor, 1992; 28S region); 12S mi-
tochondrial rDNA: 5’-TAATAATAAGAGCGA-
CGGGCGATGTGT-3’ (adapted from H1478
of Kocher et al., 1989 using sequence data for
Drosophila yakuba Burla (Clary & Wolsten-
holme, 1985)) and 5’-ТААТААААААСТАСС-
ATTAGATACCCTATTAT-3’ (adapted from
L1091 of Kocher et al., 1989); RAPD primer
А-02: (5’-TGCCGAGCTG-3’; Operon Tech-
nologies, Inc., Alameda, CA). Rationales for
primer choices are discussed in White (1994)
and White et al. (1994).
Thirty-four amplification cycles were per-
formed (1 min at 93°C, 1 min at 50°C, and 2
min at 72°C) followed by one cycle with in-
creased extension time (9 min). For RAPD
PCR, 45 amplification cycles of 1-min dena-
turation at 94°C, 1-min reannealing at 36°C,
and 2-min extension at 72°C were per-
formed. Reaction products were assayed
on 0.8-2.0% agarose minigels stained with
ethidium bromide.
Restriction Enzyme Digestion. Restriction en-
zyme digests were performed in 10- to 20-ul
reaction volumes consisting of 8-12 ul of PCR
product, 5-15 units of restriction enzyme, and
the manufacturer-supplied buffer at a final
concentration of 1x. Digests were conducted
at the manufacturer-recommended tempera-
ture (usually 37°C) for 4-48 hrs. Restriction
fragments and uncut PCR products were as-
sayed on 2.0%-agarose gels stained with
ethidium bromide. Efforts to separate poorly-
resolved fragments with 6-10% polyacryl-
amide or 2-4% MetaPhor high-resolution
agarose met with limited success and were
ultimately abandoned.
RESULTS
Key to the Unionids of French Creek
The following key was developed for iden-
tification of French Creek unionid glochidia.
One proceeds through the key by amplifying
the genomic region indicated in bold text,
digesting the PCR product with the restriction
enzyme listed after the x, and assigning a let-
ter to the resulting restriction fragment pattern
(by referring to the accompanying figure
and/or to the fragment size data in Appendix
1). Assaying undigested PCR products along-
side digested products facilitates pattern in-
terpretation and is highly recommended. Su-
perscripts refer to notes that follow the key.
While the key likely reflects phylogeny to
some extent, the data from which it was con-
structed are insufficient for testing specific
hypotheses about relationships; thus, the key
should be considered artificial.
1. ITS-1 x Mspl (Fig. 2)
Ayal AN 2
Bie Re eee pe Ligumia nasuta (Say)
CG . wee A 9°
Die En: Amblema plicata (Say)°
EME ARE Quadrula cylindrica (Say)
EXA LO en con 12
EA A Strophitus undulatus (Say)?
LEE ES Alasmidonta marginata Say
2. ITS-1 x Sau96l (Fig. 3)
A ER ER ee 3
Becks Da ER PER 8
3. 12S x Haelll (Fig. 4)
RE о aros 4
B .... Actinonaias ligamentina (Lamarck)
CRIME Lampsilis siliquoidea (Barnes)?
4. ITS 1-2 x Mspl' (Fig. 5)
A DEN TS DR ORNE 5
ss ss see oso o т
BR rare Lampsilis fasciola Rafinesque
5. 12S x Rsal (Fig. 6)
PRA O EE Villosa iris (1. Lea)
Ds Epioblasma Spp: 1: 69
6. ITS 1-2 x Mboll (Fig. 7)
А Epioblasma torulosa rangiana
(1. Lea)"
A" ....Epioblasma triquetra (Rafinesque)
7. 11$-1 x Aval (Fig. 8)
Ще Lampsilis cardium Rafinesque,
Lampsilis ovata (Say)'
(CR RS eee Ligumia recta (Lamarck)
8. ITS-1 x Accl (Fig. 9)
IEA ee Ptychobranchus fasciolaris
(Rafinesque)
DAS ET Villosa fabalis (1. Lea)
9. ITS-1 x BstEll (Fig. 10)
LOS, Зе Elliptio dilatata (Rafinesque)
D: rate A 10
10. ITS 1-2 x Mspl' (Fig. 11)
Bette ое 11
A Ame A Fusconaia subrotunda (|. Lea)
11. RAPD A-02 (Fig. 12)
Ar Pleurobema clava (Lamarck)"
В ...Pleurobema sintoxia [= coccineum]
(Rafinesque)
12. ITS-1 x BamHI (Fig. 13)
Ne. ee Lasmigona costata (Rafinesque)
OIR AR : 13
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 187
13. ITS-1 x Hinfl (Fig. 14) OA CE EN N RE 15
À ...Anodontoides ferussacianus (|. Lea) 15. ITS 1-2 x Mspl' (Fig. 16)
ОН ome RCE EVE AP 14 SERRE Pyganodon [= Anodonta] grandis
14. ITS-1 x Mboll (Fig. 15) (Say)
E de Lasmigona compressa (l. Lea) AA Lasmigona complanata (Barnes)
1500
-600
-100
a. uneut 71 115111190 11111511 1120 ı 1125
À 1500
= -600
00
¡A E DE—F—GH Pattern
E ze | ———+P—'Am'— An — Tribe
FIG. 2. ITS-1 PCR products from 25 French Creek unionid species digested with Mspl. Restriction fragment
patterns (A-H) separate species into their respective tribes. Tribe Am = Amblemini, An = (subfamily) An-
odontinae, L = Lampsilini, Р = Pleurobemini. Tribe Lampsilini (patterns A, В): 1 = Epioblasma torulosa
rangiana, 2 = Epioblasma triquetra, 3 = Lampsilis cardium, 4 = Lampsilis fasciola, 5 = Lampsilis ovata, 6 =
Lampsilis siliquoidea, 7 = Villosa fabalis, 8 = Villosa iris, 9 = Actinonaias ligamentina, 10 = Ptychobranchus
fasciolaris, 11 = Ligumia recta, 12 = Ligumia nasuta; tribe Pleurobemini (pattern C): 13 = Elliptio dilatata, 14
= Pleurobema clava, 15 = Pleurobema sintoxia, 16 = Fusconaia subrotunda; tribe Amblemini (patterns D, E):
17 = Amblema plicata, 18 = Quadrula cylindrica; subfamily Anodontinae (patterns F, G, H): 19 = Anodontoides
ferussacianus, 20 = Pyganodon grandis, 21 = Lasmigona compressa, 22 = Lasmigona costata, 23 = Las-
migona complanata, 24 = Strophitus undulatus, 25 = Alasmidonta marginata. Tribe designations follow
Vaught (1989). Gels shown throughout key are 2.0% agarose. Size marker used throughout key is 100-bp
ladder.
188
WHITE, MCPHERON 8 STAUFFER
-100
a. uncut 1 2 3 4 9 6 7 8 9 10 11
100
eo ah |
6. Sau961-cut
FIG. 3. ITS-1 PCR products from ‘‘1-A”’ species digested with Sau96l. 1 = Epioblasma torulosa rangiana,
2 = Epioblasma triquetra, 3 = Actinonaias ligamentina, 4 = Lampsilis cardium, 5 = Lampsilis fasciola, 6 =
Lampsilis ovata, 7 = Lampsilis siliquoidea, 8 = Ligumia recta, 9 = Villosa iris, 10 = Villosa fabalis, 11 =
Ptychobranchus fasciolaris.
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 189
a. uncut
> "PA
b. Haelll-cut
1500
-600
-100
12s 4.75) 7 00 9
-1500
-600
-100
B C
FIG. 4. 12S PCR products from “2-А” species digested with Haelll. 1 = Lampsilis cardium, 2 = Lampsilis
fasciola, 3 = Lampsilis ovata, 4 = Ligumia recta, 5 = Epioblasma torulosa rangiana, 6 = Epioblasma triquetra,
7 = Villosa iris, 8 = Actinonaias ligamentina, 9 = Lampsilis siliquoidea.
Notes to Accompany the Key
“Includes Ptychobranchus fasciolaris, in
contradiction to White et al., 1994; the spec-
imen identified in White et al. (1994) as P.
fasciolaris is almost certainly Elliptio dilatata.
PThe Pleurobema sintoxia specimen from
Foster Corner exhibited a unique pattern (Fig.
12):
“One ofthe 18 Amblema plicata specimens
from the lower Muskingum River, Ohio, ex-
hibited a unique pattern quite similar to that
of Ligumia nasuta (Fig. 18).
In contradiction to White et al., 1994; the
specimen identified by White et al. (1994) as
Strophitus undulatus was subsequently re-
identified as Pyganodon grandis by A. E.
Bogan.
°Two Lampsilis siliquoidea specimens from
the French Creek drainage (one of the three
190 WHITE, MCPHERON & STAUFFER
— uncut — р Mspl-cut ——
1234567
172.34 5,637
-100
FIG. 5. ITS 1-2 PCR products from ““3-A” species digested with Mspl. 1 = Epioblasma torulosa rangiana,
2 = Epioblasma triquetra, 3 = Villosa iris, 4 = Lampsilis cardium, 5 = Lampsilis ovata, 6 = Ligumia recta, 7
= Lampsilis fasciola.
from Venango and the one from Conneaut
Outlet) exhibited patterns with three bands
instead of two (Fig. 19).
'A and A’ are most reliably distinguished by
digesting samples of known DNA and assay-
ing them in lanes adjacent to the unknown
DNA. Digesting several samples of each
known and unknown DNA is recommended,
as it allows one to intersperse samples of
each type on a single gel for easier detection
of subtle length differences. Assays should
be run on at least a 2%-agarose gel, for as
long as possible, to achieve maximal sepa-
ration.
°Couplet 6 reliably separates two of the
four Epioblasma torulosa rangiana speci-
mens examined (one of the two from
Venango and the one from Utica) from the
three Epioblasma triquetra specimens exam-
ined. The broader utility of this couplet is un-
certain; it should be used with caution. Also
see note f.
"federally endangered species
'Lampsilis cardium and Lampsilis ovata
specimens could not be distinguished from
each other using any of the primers and re-
striction enzymes tried (White, 1994: appen-
dix B2). It is conceivable that these species
hybridize in French Creek; some specimens
exhibited intermediate shell morphologies
and could not be identified to species with
certainty on the basis of external characters
(A. E. Bogan, pers. comm.).
Reliability of the Key
The key was tested extensively using adult
unionids identified morphologically. In its an-
notated form, it proved valid for all French
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 191
Rsal-
uncut -— cut —
122535 1,273
-100
ET
0 A
FIG. 6. 12S PCR products from ‘‘4-A” species di-
gested with Rsal. 1 = Epioblasma torulosa rangi-
ana, 2 = Epioblasma triquetra, 3 = Villosa iris.
Creek specimens examined. It was also valid
for all Ohio and West Virginia A. plicata (Fig.
20), L. siliquoidea, and L. costata specimens
examined. Moreover, glochidia obtained
from a gravid French Creek female L. costata
followed the key, exhibiting restriction frag-
ment patterns identical to those of adult L.
costata specimens, as expected (data not
shown).
Identification of Unknown Glochidia with
the Key
Four unknown glochidia from the gills of a
tippecanoe darter (Etheostoma tippecanoe
Jordan & Evermann) collected 20 July 1993 in
French Creek downstream of Utica, Pennsyl-
vania, exhibited restriction fragment patterns
identicalto those of adult V. fabalis specimens
(unpubl. data). In a larger-scale test of the
technique's utility, all glochidia found on
fishes collected 6 June 1994 at the LeBoeuf
Creek site were analyzed. Of the 115 glochidia
Mbo ll -
г uncut cut—
12321253
100
1
ALA?
FIG. 7. ITS 1-2 PCR products from “5-0” species
digested with Mboll. 1 = Epioblasma torulosa ran-
giana, 2 = Epioblasma triquetra, 3 = E. triquetra.
r— uncut — — Aval-cut—
т 2, 53
1500
-600
100
LA — |
0
FIG. 8. ITS-1 PCR products from “‘4-А”’ species
digested with Aval. 1 = Lampsilis cardium, 2 =
Lampsilis ovata, 3 = Ligumia recta.
192 WHITE, MCPHERON & STAUFFER
Accl-
с uncut г cut —
122 al a2
-1500
-600
-100
eet
A 0
FIG. 9. ITS-1 PCR products from ‘‘2-B” species
digested with Accl. 1 = Ptychobranchus fasciolaris,
2 = Villosa fabalis.
processed, 72 (63%) were amplified success-
fully (i.e., their ITS-1 PCR products were vis-
ible on an agarose gel stained with ethidium
bromide). Of these, 66 (92%) were identifi-
able; the other six yielded restriction frag-
ments too faint to be seen. Fourteen of the 43
glochidia not amplified successfully were in
the first set of samples, extracted using a pro-
tocol that differed slightly from that ultimately
adopted. Disregarding this flawed first at-
tempt, the amplification success rate was 72
out of 102 (71%).
All 66 glochidia identified exhibited the
restriction fragment patterns characteristic
of Ptychobranchus fasciolaris (Fig. 21), a
species for which no hosts are currently
known (Hoggarth, 1992). Four Etheostoma
blennioides Rafinesque, three Etheostoma
flabellare Rafinesque, five Etheostoma ni-
grum Rafinesque, and one Etheostoma zon-
ale (Cope) harbored the glochidia. These four
darter species are therefore suggested ten-
tatively to be P. fasciolaris hosts, pending
verification through laboratory inoculation
studies.
Bst Ell-
r— uncut — -— cut —
1,2, 8 4 1727 394
-100
ge vee N
A 0
FIG. 10. ITS-1PCR products from “1-С” species
digested with BstEll. 1 = Elliptio dilatata, 2 =
Fusconaia subrotunda, 3 = Pleurobema clava, 4 =
Pleurobema sintoxia.
DISCUSSION
Distinguishing Unionid DNA from Host Fish
DNA
Without exception, the ITS-1 regions of the
fishes examined are markedly different in
length from those of the unionids. For single
individuals of five of the six darter species
examined (E. blennioides, E. flabellare, E. tip-
pecanoe, Etheostoma variatum Kirtland, and
E. zonale), the product is approximately 690-
710 bp; for the sixth darter, Etheostoma
maculatum Kirtland, the product is approxi-
mately 410 bp long (White, 1994: Fig. 2.3).
Among most of the unionids, the ITS-1 prod-
uct ranges from approximately 580 to 625
bp; for Alasmidonta marginata Say and Stro-
phitus undulatus (Say), it is approximately
950-1,050 bp long (see uncut products in
Fig. 2). Because the length ranges for fishes
and unionids are non-overlapping, any host-
fish DNA contaminating glochidial DNA is
easily recognized as such. Furthermore,
when ITS-1 PCR products of the six darter
specimens are digested with Mspl, they yield
restriction fragment patterns different from all
unionid patterns. Hence, even if the glochid-
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 193
Mspl-
> ÚNCUL + CU
1232, Sd Al
2 3 4
100
E. A rt A ЗЕЕ
FIG. 11. ITS 1-2 PCR products from “9-0” species digested with Mspl. 1 = Pleurobema clava, 2 =
Pleurobema sintoxia, 3 = Fusconaia subrotunda, 4 = F. subrotunda.
ial identification method described herein
were applied to fishes (e.g., salmonids)
whose ITS-1 regions are close to the union-
ids’ in length (Pleyte et al., 1992), contamina-
tion could be detected reliably by digesting
the host fish’s ITS-1 product and assaying it
alongside the digested products of the
glochidia it harbored. The contaminating
DNA could be factored out of the RFLP anal-
yses by disregarding restriction fragments
present in both gel lanes.
Current Limitations of the Technique
Identifying glochidia on naturally infected
fishes is a hit-or-miss approach to discover-
ing hosts of a particular unionid species of
interest. To maximize the chances of suc-
ceeding, it is important to collect fishes from
sites where the unionid species of interest is
abundant relative to other species (or at least
where it is abundant relative to other sites).
As the preliminary LeBoeuf Creek study
12 3 4 5 6
и 1500
100
ges B
FIG. 12. RAPD A-02 PCR products from ‘‘10-A”’
species. 1-3 = Pleurobema clava, 4-6 = Pleu-
robema sintoxia.
194 WHITE, MCPHERON 8 STAUFFER
-—— uncut ——— -— BamHl-cut —
SS
123 4 51
—100
FIG. 13. ITS-1 PCR products from “1-Е” species digested with BamHI. 1 = Anodontoides ferussacianus,
2 = Pyganodon grandis, 3 = Lasmigona complanata, 4 = Lasmigona compressa, 5 = Lasmigona costata.
я 100
+ AM a
Е
FIG. 14. ITS-1 PCR products from ‘12-0’ species
digested with Hinfl. 1 = Anodontoides ferussa-
cianus, 2 = Pyganodon grandis, 3 = Lasmigona
complanata, 4 = Lasmigona compressa.
(— uncut — — Mboll-cut -
ı 2 ge
1500
— №
00
|
О
FIG. 15. ITS-1 PCR products from ‘‘13-B”’ species
digested with Mboll. 1 = Pyganodon grandis, 2 =
Lasmigona complanata, 3 = Lasmigona com-
pressa.
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 195
CU nn МЕС
1210445, 001.2%3:4,5:6
100
FIG. 16. ITS 1-2 PCR products from ‘‘14-0” species digested with Mspl. 1-3, 5, 6 = Pyganodon grandis;
4 = Lasmigona complanata.
au
‚uncut reut-,
| -100
12
1 2
FIG. 17. ITS-1 PCR products from anomalous and
Standard Pleurobema sintoxia specimens digested
with Mspl. 1 = anomalous pattern, 2 = standard
pattern.
Mspl-
r uncut — — cut —
12 200 573
FIG. 18. ITS-1 PCR products from anomalous and
standard Amblema plicata specimens digested
with Mspl. 1 = Ligumia nasuta, 2 = anomalous Am-
blema plicata, 3 = standard A. plicata.
196 WHITE, MCPHERON 8 STAUFFER
Haelll-
uncut- — cut
-100
he 02 17:2
FIG. 19. 12S PCR products from anomalous Lamp-
silis siliquoidea specimens digested with Haelll.
Specimens exhibit 3-banded pattern instead of
standard 2-banded pattern.
demonstrated, this will not guarantee suc-
cess, however. Additionally, fishes should be
collected repeatedly throughout the full du-
ration of the unionid’s breeding period.
The glochidial amplification procedure cur-
rently has a success rate below 100%. Most
unsuccessful amplification attempts were
likely the result of glochidia being lost during
transfer from host to extraction buffer; once
excised from the host, glochidia are ex-
tremely difficult to see. Improvements in the
transfer technique could increase the ampli-
fication success rate dramatically. The iden-
tification success rate, already quite high,
could probably be increased by gel-purifying
and reamplifying very faint PCR products
prior to restriction enzyme digestion.
Extending the Key Beyond French Creek
To apply the method to an aquatic system
other than the French Creek drainage, some
preliminary work is required. First, tissue
samples must be obtained from several indi-
viduals of each unionid species found in the
study system. Ideally, each species should
be represented by specimens collected at a
variety of sites.
Next, the reliability of the key, for the
study-system species included in it must be
assessed. All specimens of each such spe-
cies should be analyzed using the key, to see
whether they yield the expected restriction
fragment patterns for each enzyme (as did
the West Virginia and Ohio specimens we ex-
amined). If they do not, the key will have to be
modified accordingly.
The key will also have to be extended to
include any study-system species not found
in French Creek. This is most easily accom-
plished as follows: first, analyze a single
specimen of each new species, using the
French Creek key. If a specimen yields a
novel restriction fragment pattern for a cer-
tain couplet, test all individuals of the species
to see if they share the pattern; if they do,
modify the key accordingly. If a specimen
yields no novel patterns, proving indistin-
guishable from a species already included in
the key (or from another new species), screen
single individuals of the indistinguishable
species pair (or group) with a variety of prim-
ers and restriction enzymes until a diagnostic
difference is found. (Consulting Appendix B
of White, 1994, might prove useful in this re-
gard.) Alternately, sequence a moderately
variable region of the genome of each spe-
cies and scan the sequence data for restric-
tion site differences. Finally, verify that the
differences found apply to all individuals of
the species, then modify the key accordingly.
Publish modified versions of the key
promptly to save other investigators precious
time and resources.
Overall Assessment of the Technique
Using a molecular genetic key to identify
glochidia attached to fishes has distinct ad-
vantages over traditional means of identifying
putative unionid hosts (White et al., 1994).
The laboratory procedures are relatively fast
and easy to perform. Once a key has been
developed, glochidia can be identified in one
or two days; the techniques involved can be
learned (if not mastered) in a week. The
method is also relatively inexpensive, partic-
ularly if one has access to a laboratory al-
ready equipped for molecular genetic re-
search (see White, 1994: appendix C, for cost
analysis).
The data generated to develop keys are
potentially valuable to unionid systematists,
197
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS
-100
а. ие 4ı1ıı5 1 ı1ıı (O tt 15
b. Mspl-cut
FIG. 20. ITS-1 PCR products from 15 Amblema plicata specimens from three drainages, digested with
Mspl. 1-5 = French Creek specimens, 6-10 = Dunkard Creek specimens, 11-15 = Muskingum River
specimens.
198 WHITE, MCPHERON & STAUFFER
Mspl- Sau96l- Accl-
Г uncut 7 cut — cut cut —
1 at 2
-100
2 Ae
FIG. 21. ITS-1 PCR products from LeBoeuf Creek glochidium and adult Ptychobranchus fasciolaris, di-
gested with Mspl, Sau96l, and Accl. The glochidium exhibits restriction fragment patterns identical to those
of the adult P. fasciolaris. The glochidium was removed from the gills of an Etheostoma flabellare specimen.
1 = glochidium, 2 = adult P. fasciolaris.
as well. For example, the RFLP analysis of
ITS-1 shown in Figure 2 suggests that pat-
terns of site gain and loss could demarcate
tribal boundaries. In many organisms, this
sort of information has been used to recon-
struct phylogenetic relationships (reviewed in
Avise, 1994). Our study was not designed to
provide the complete matrix necessary to an-
alyze this question, but our data (Summarized
in Appendix 2) do provide a starting point for
systematists wishing to pursue the issue of
higher relationships. (Note that many of the
results presented in Appendix 2 are unrepli-
cated and/or based on small numbers of
specimens.)
The method is well suited to conservation
work. It does not entail killing adult unionids
and hence can be used with endangered
species. It yields results that are relevant to
natural communities. It can even furnish in-
sights into subtle ecological matters, such as
patterns of host-fish partitioning among
unionids. Finally, it can be applied to diverse
systems with large numbers of fish and
unionid species.
ACKNOWLEDGMENTS
This material is based upon work sup-
ported under a National Science Foundation
Graduate Fellowship. Any opinions, findings,
conclusions, or recommendations expressed
in this publication are those of the authors
and do not necessarily reflect the views of
the National Science Foundation. This re-
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 199
search was also supported by a grant to Jay
R. Stauffer, Jr., from the Pennsylvania Wild
Resources Conservation Fund. G. M. Davis
and À. E. Bogan encouraged us to pursue the
research and provided valuable feedback
throughout the project. M. E. Gordon, M. C.
Hove, R. J. Neves, C. Saylor, W. C. Starnes,
and J. D. Williams contributed important in-
sights. C. Bier, À. E. Bogan, and G. T. Wat-
ters assisted with specimen collection and
identification. Field assistance was also pro-
vided by N. J. Bowers, K. L. Bryan, C.
Gatenby, M. J. Gutowski, E. À. Hale, K. A.
Kellogg, J. A. Lee, T. Proch, R. Shema, G. A.
Smith, T. D. Stecko, and E. S. vanSnik. J.
Clayton provided access to West Virginia col-
lection sites; C. Copeyon assisted in obtain-
ing endangered-species permits. N. J. Bow-
ers and W. S. Sheppard provided primer and
restriction enzyme samples. Laboratory as-
sistance and advice were furnished by N. J.
Bowers, E. Carlini, C. L. Crego, D. Cox-Fos-
ter, and H.-Y. Han.
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Revised Ms. accepted 20 May 1996
MOLECULAR GENETIC IDENTIFICATION OF UNIONIDS 201
APPENDIX 1. Estimated sizes of restriction fragments used in the key (excluding fragments shorter than
100 bp).
Couplet Pattern Fragment Size (bp) Couplet Pattern Fragment Size (bp)
1 = (275 — 285) + 185 5 = 415
Be 305 + 185 A= 250 + 180
C= 275 + 140* 6 A= 575 + 280 + 205
D= 305 + 140** А’ = 575 + 290 + 195
EE 465 + 140 7 0= 615
Me (495 — 505) + 140 = 510
G= 960 8 0/= 575
H = 895 А = 370 + 225
*anomalous P. sintoxia = 9 = 565
205 + 140 A= 340 + 225
*anomalous A. plicata = 10 A= 385 + 270 + 195
305 + 170 A= 400 + 275 + 195
2 A= 350 + 230 11 = 585
Be 240 + 225 + 185 = 555
3 А = 240 + 165 12 — (590 — 620)
B = 200 + 165 = 430 + 190
c= 235 + 195* 13 = 455
*anomalous L. siliquoidea = B = 520
= 245 + 205 + 170 14 = (600 — 610)
2= 275 + 245 + 200 = 370 + 240
4 A= 390 + 270 + 170 15 A= 500 + (445 — 460) +
he 375 + 265 + 170 195
B= 310 + 265 + 170 А’ = 515 + 460 + 195
WHITE, MCPHERON 8 STAUFFER
202
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MALACOLOGIA, 1996, 38(1-2): 203-212
QUANTITATIVELY SAMPLING LAND-SNAIL SPECIES RICHNESS IN
MADAGASCAN RAINFORESTS
Kenneth C. Emberton, Timothy A. Pearce 8 Roger Randalana
Molluscan Biodiversity Institute, 216-A Haddon Hills, Haddonfield, New Jersey 08033, U.S.A.
and Institute for the Conservation of Tropical Environments, B.P. 3715, Tsimbazaza,
Antananarivo 101, Madagascar
ABSTRACT
Land-snail species richness in tropical rainforests tends to be high but difficult to assess
because of low densities and often small shell sizes. We tested three quantitative sampling
methods in primary rainforests of southeastern Madagascar. Timed searching yielded seven
times aS many micro-snail species (Species that during at least part of their life have shells < 5
mm maximum dimension) per person-hour as either litter sampling or soil-plus-litter sampling.
The number of species found in 20 m * 20 m during three person-hours of searching, however,
was boosted a maximum of 38% by one eight-liter sample each of litter and soil-plus-litter.
Litter sampling and timed searching both yielded more than 1.5 times the proportion of live-
collected species as soil-plus-litter sampling. Sampling method was unbiased toward 12 of the
20 commonest species, but three large, presumed arboreal species were favored by timed
searches; two minute, presumed burrowers by soil-plus-litter sampling; and three minute,
cryptically colored species by both litter and soil-plus-litter sampling. A 1.2-mm sieve caught
at least 78% of the total specimens and passed adults of 7% of species, of which the smallest
adult dimension was 1.0 mm. These results suggest that the best sampling strategy is timed
searching for micro-snails, while incidentally collecting macro-snails and litter-plus-soil for later
picking of the 5.5-1.2 mm and the 1.2-0.85 mm, dry-sieved fractions. This strategy should be
transferable to other tropical-rainforest land-snail faunas.
Key words: Gastropoda, tropical biodiversity, leaf-litter biota, soil biota.
INTRODUCTION
Land snail faunas of tropical rainforests
tend to be quite diverse (maximum reported:
52 species per 4 ha) despite often low den-
sities (Emberton, 1995a [and citations there-
in]; Tattersfield, 1994, in prep.; F. Thompson,
pers. commun.; despite Solem’s [1984] un-
documented statement to the contrary). Much
of this diversity consists of micro-gastropods
(< 5 mm greatest dimension), the collection
of which can be extremely labor-intensive
(Emberton, 1994, 1995a, 1996; DeWinter,
1995; F. Thompson, pers. commun.; P. Tat-
tersfield, pers. commun.; R. Ramirez, pers.
commun.). Because most tropical rainforests
are vastly undercollected for micro-gastro-
pods and are undergoing irreversible defor-
estation, great urgency attaches to collecting
these mostly undiscovered, undescribed mol-
luscs as efficiently and thoroughly as pos-
sible. Because of the prime importance in
land-snail systematics of preserving anato-
mies and DNA in ethanol, sampling methods
should maximize live collections. Because
land snails are generally so patchily distrib-
203
uted, even within seemingly uniform forest,
sampling should probably avoid random-
quadrat methods (Emberton, 1995a).
Timed searches by experienced collectors
are a well-proven method of quantitatively
sampling patchily distributed organisms (Cod-
dington et al., 1991). One of us has recently
advocated timed searches as the most effi-
cient collecting method for tropical rainforest
micro-snails (Emberton, 1995a), and has ap-
plied such data toward assessing conserva-
tion priorities (Emberton, 1996). The efficacy
of timed searches for collecting all or a sub-
stantial portion of the micro-gastropod fauna,
however, has never been tested, to our knowl-
edge.
Collection of measured quantities of se-
lected leaf litter is another quantitative sam-
pling method that has proven effective for
tropical-rainforest land-snail communities
(Tattersfield, 1994). Soil-plus-litter samples
also often yield species that are collected in
no other way (F. Thompson, 1995). Some
species may be soil specialists, other species
may take refuge in soil from drying litter, and
soil can accumulate dead shells of litter spe-
204 EMBERTON, PEARCE & RANDALANA
cialists (pers. observ.; Burch & Pearce, 1990).
Processing of soil-plus-litter samples, how-
ever, is more labor-intensive than processing
of litter samples.
The purpose of this paper is to compare
the performances of (a) timed searching, (b)
litter sampling, and (c) soil-plus-litter sam-
pling for determining the species richness of
and obtaining live material of the micro-land-
snail fauna of Madagascan rainforests, and
to arrive thereby at the most efficient overall
sampling strategy.
METHODS AND MATERIALS
We sampled 48 plots, each 20 m x 20 m, at
16 stations on three widely separated moun-
tains in southeastern Madagascar (Fig. 1, Ta-
ble 1). Localities and stations were chosen to
serve both for this study and for testing di-
versity patterns between the Vohimena and
Anosy mountain chains (Emberton, 1996,
Emberton et al., in review). Stations were at
100 m elevation intervals from 100 m to 500
m and at 200 m elevation intervals above 500
m, with a station at the highest or a local
summit.
Stations were restricted to primary forest
that had no more than limited selective cut-
ting. For each station, we recorded the ele-
vation (average of two Thommen Altitrek al-
timeters, calibrated from topographic maps),
latitude and longitude (from topographic
maps), and the topography (Summit, ridge,
slope, or valley). For more extensive data on
these stations, see Emberton (in review).
At each station, we sampled three adjacent
20 m x 20 m replicate plots, each marked off
with flagging tape. We sampled 25 January
to 7 February 1995, during the rainy season,
within one week of heavy rains, when snails
and slugs seemed likely to be most active
and therefore perhaps easier to find. We in-
cluded only micro-snails, which for the pur-
poses of this study we defined as those spe-
cies that during at least part of their life have
shells that are smaller than 5 mm maximum
dimension (the vast majority remain below
this size as adults).
Timed searching was for three person-
hours per plot: one-half hour by six collec-
tors. Three of these collectors (RR and two
assistants who had been trained by all three
authors) were constant over all stations and
plots, and the other three were hired locally
and trained by RR. As incentives, small cash
prizes were offered for the most snails and
the smallest snail collected in each plot. Mi-
cro-molluscs were hand-collected into
30-ml, snap-cap vials, drowned overnight,
then fixed and preserved in 70-90% ethanol.
Litter samples and soil-plus-litter samples
were each eight | in volume per plot, col-
lected over a 30-minute period by KCE and
TAP, respectively. Both types of sampling
were from moist, sheltered microhabitats
such as beside logs, between buttress roots
of trees, within Asplenium and Pandanus ro-
settes, under and near piles of Ravenala and
palm fronds, and in moist depressions (Em-
berton & Arijaona, in press: fig. 2). Litter and
litter-plus-soil samples were collected into
four-mill plastic bags and kept as cool as
possible until processing, a maximum of
three days later, with daily opening of each
bag for aeration.
All litter and soil-plus-litter samples were
wet-sieved through three mesh sizes: 11.5
mm, 5.5 mm, and 1.2 mm. We used wet siev-
ing (i.e. washing the samples with water) in
order to process quickly samples wet from
recent or current rains, and to assure live re-
covery of slugs, semislugs, and thin-shelled
species. Sieve boxes for the first three size
fractions consisted of large plastic storage
boxes (55 x 48 x 35 cm) from which the bot-
toms had been cut (leaving a 3.8-cm margin),
then covered with hardware cloth (11.5 mm),
hardware mesh (5.5 mm), or hardware screen
(1.2 mm) (the latter two supported by hard-
ware cloth) held in place with duct tape. The
three sieve boxes were nested over an intact
box to catch effluent during washing of a lit-
ter sample and were transferred to a second
box if the first filled. Whenever the litter or
soil-plus-litter samples were not too wet, as
much dry-sieving as possible was performed
prior to wet-sieving. The first two sieve frac-
tions were picked immediately for all inverte-
brates by the authors, aided by teams of local
workers, each of whom was carefully trained
and monitored by at least one of the authors.
The third fractions (retained by the 1.2-mm
sieve) were fixed and stored for no longer
than three weeks in an equal or greater vol-
ume of 90% ethanol (the resulting ethanol
concentration averaged about 60%). The ef-
fluent was caught by pouring all sieved wash
water from the bottom box or boxes through
two nested nylon stockings, from which ex-
cess water was squeezed gently, then which
were fixed and stored in an equal or greater
volume of 90% ethanol.
RAINFOREST LAND-SNAIL SAMPLING 205
47°30
ILAPIRY
TOLAGNARO
VASIHA
FIG. 1. The three mountains sampled in the Anosy and Vohimena chains, southeastern-most Madagascar
(see inset). Contours are shown at 500 m and 1,000 m. The dashed line indicates Andohahela Reserve. The
dot indicates the city of Fort Dauphin (= Tolagnaro).
All > 1.2-mm sieve fractions were picked
for all invertebrates by RR and six assistants,
each of whom was trained by all three au-
thors and monitored by RR. Non-molluscan
invertebrates are being distributed among in-
terested specialists. Only molluscs are anal-
ysed in this paper.
To test the efficiency of the 1.2-mm sieve
at catching snails, the sieving effluent (i.e., all
that passed through the 1.2-mm sieve) from
one plot per station (the plot whose upper
sieve fractions yielded the greatest number
of species) was further sieved through U.S.A.
Standard Testing Sieves Nos. 20 and 30
(0.85 mm and 0.60 mm). Both these fine frac-
tions were picked for snails and shells by RR
and four trained, monitored assistants, wear-
ing Optivisor magnifying lenses of 2x magni-
fication. Picking of all sieve fractions was
performed on a white or light-gray, hard sur-
face. Those snails from the < 1.2 mm fraction
were used only for testing the sieve effi-
206 EMBERTON, PEARCE & RANDALANA
TABLE 1. Stations sampled for land snails in southeastern Madagascar. Elv = elevation in
meters, r/s/v = ridge, slope, and valley.
# Mountain Elv Latit. S Long. E Topogr
1 Mahermano 340 24.26.12 47.13.13 summit
2 Mahermano 300 24.26.17 47.13.10 slope
3 Mahermano 200 24.26.15 47.13.04 slope
4 Mahermano 100 24.26.22 47.12.41 valley
5 llapiry 540 24.51.40 47.00.20 summit
6 llapiry 500 24.51.33 47.00.27 ridge
te llapiry 400 24.51.27 47.00.38 r/s/v
8 llapiry 300 24.51.36 47.00.40 slope
9 llapiry 200 24.51.39 47.00.46 slope
10 Vasiha 860 24.55.18 46.44.19 summit
11 Vasiha 700 24.55.23 46.44.27 slope
12 Vasiha 500 24.55.19 46.44.45 slope
13 Vasiha 400 24:55:25 46.44.45 valley
14 Vasiha 300 24.55.37 46.44.49 slope
15 Vasiha 200 24.56.13 46.45.13 slope
16 Vasiha 100 24.56.20 46.46.07 slope
ciency, and were not included in the main
data matrix or data analysis.
All snails and shells were sorted and iden-
tified to morphospecies by KCE. For each
morphospecies, a relatively intact adult rep-
resentative was chosen and was photo-
graphed in two to five diagnostic views at
standard magnifications, using a Polaroid
camera mounted on a Wild dissecting micro-
scope. The resulting reference collection and
file of photographs were used to identify all
specimens, both adults and juveniles, except
for some juveniles of the most minute sieve
fractions, which were identified only to genus
or family. Systematic treatments of the mor-
phospecies, 85% of which are new, are in
progress; vouchers are in the collection of
the Molluscan Biodiversity Institute, with
types and references to be placed in the
Madagascar national museum (Parc Bota-
nique et Zoologique de Tsimbazaza, An-
tananarivo) and in the Academy of Natural
Sciences of Philadelphia. (Patterns of diver-
sity, distribution, and abundance of the mor-
phospecies are treated in a separate paper
[Emberton, in review].)
To compare efficiencies of the three meth-
ods, we calculated the number of person-
hours required to collect and—in the case of
litter and litter-plus-soil—to wet-sieve and to
pick an average sample (for all invertebrates).
We then computed the mean numbers of
molluscan specimens and of species ob-
tained per person hour by each method. We
were not able to calculate the percent of
picking time devoted to molluscs alone, so
our person-hour calculations were overesti-
mates.
We used analysis of variance (ANOVA) by
least-squares estimation (Wilkinson, 1990) to
evaluate differences among the three sam-
pling methods in (a) number of species col-
lected per plot, (b) percent of the total spe-
cies that were found in each plot, and (c)
percent of species collected live. For the per-
cent of the total species collected within
each plot, we used the entire data set in a
one-way ANOVA. For species number and
percent live, however, we factored out the
effects of locality (mountain) and elevation by
including them in a three-way ANOVA on the
largest possible subset of the data including
all three mountains (see Emberton et al., in
review: fig. 2), which had to be limited to 200
m and 300 m elevations (Table 1).
For each species representing at least one
percent of the total specimens, we used chi-
square analysis to test among the three sam-
pling methods for equal numbers of speci-
mens. Predicted frequencies were based on
the total number of specimens resulting from
each method. Probability estimates were
Bonferroni-adjusted to allow for multiple
tests.
RESULTS
Including the macro-snail species that
showed up in the upper sieve fractions, we
collected a total of 87 species (also see be-
low). Taxonomically, these species were dis-
RAINFOREST LAND-SNAIL SAMPLING
207
TABLE 2. Average time investments and productivities of three sampling methods. Collect = collecting
within a 20 m x m plot, Sieve = wet sieving of an eight-liter sample from a 20 m x 20 m plot, Pick =
picking all invertebrates (not just gastropods) from the > 1.2-mm sieved sample, Total hours = total
person-hours per plot sample, Specm./p-hr = mean number of specimens obtained per person hour,
Spp./p-hour = mean number of species obtained per person hour, Spp./specm. = proportion of mean
species to mean specimens.
Person-Hours per Task
Total Specm. Spp./ Spp./
Method Collect Sieve Pick hours p-hr p-hr specm
Timed search 3.0 0.0 0.0 3.0 9.36 3.03 0.32
Litter sample 0.5 4.8 4.7 10.0 0.88 0.46 0.52
Soil-plus-litter 0.5 4.8 9.8 15.1 0.91 0.41 0.45
tributed as follows, with higher classification Infraorder HELICIDA
following Abbott 8 Boss (1989) for ‘‘Proso- Superfamily HELICARIONOIDEA
branchia” and Gymnomorpha and Nordsieck Helicarionidae: Sesarinae
(1986) for Pulmonata: FMC > ss e RE 1
Helicarionidae: Microcystinae
“Subclass PROSOBRANCHIA” u ee are, bs
Order MESOGASTROPODA Helicarionidae: Ariophantinae
Superfamily CYCLOPHOROIDEA UDOT AE SET i
Cyclophoridae _Malagarion EEE Tr. 1
Do Ne ta a ct 17 Helicarionidae: Macrochlamydinae
Cyathopoma............. 4 MVA CURAS EDI AAA 9
EAS ER 1
Diplommatinidae We excluded from analysis all specimens of
MISMA: 2 Sr мон 1 the one slug species (Veronicellidae) and of
Superfamily LITTORINOIDEA the six snail species that were considered al-
Pomatiasidae ways too large, even as juveniles, to qualify
WODIGGDROIA кие Фаня 3 as micro-molluscs (< 5 mm): the one Haine-
Superfamily RISSOOIDEA sia, two of the three Tropidophora, and all
Assimineidae three acavids.
Omphalötroßis ws we 2.0.4. 2 Distributions of the 80 analyzed species
Subclass GYMNOMORPHA among samples, totalling 2,430 specimens,
Order SOLEOLIFERA are archived at the Molluscan Biodiversity In-
Veronicellidae ............ 1 stitute (MBI) and the Academy of Natural Sci-
Subclass PULMONATA: Order ences of Philadelphia (ANSP).
STYLOMMATOPHORA The three sampling methods required
Suborder ORTHURETHRA drastically different investments of time to
Superfamily CHONDRINOIDEA acquire gastropods (Table 2). Timed search
Orculidae was by far the most efficient, yielding about
FAURE. a 2 ten times the number of specimens and
Suborder SIGMURETHRA seven times the number of species per per-
Infraorder ACHATINIDA son-hour as either litter sampling or soil-plus-
Superfamily ACHATINOIDEA litter sampling. These advantages are inflated
SUDUINIdAS. ee Pen ic 3 somewhat, however, because we took time
Superfamily STREPTAXOIDEA to pick all invertebrates.
Streptaxidae ............. 14 The litter and soil-plus-litter methods were
Superfamily ACAVOIDEA more diverse than timed search, yielding
Acavidae about half again as many species per speci-
И 85. AGA AA 1 men (also see below).
Beaver 1 Table 3 gives ANOVA results for number of
Helicophanta ............ 1 species collected per 20 m x 20 m plot. Sam-
Superfamily PUNCTOIDEA pling method had a highly significant effect
Charopidae sr. 20% 40 er 9 when the less significant effect of elevation
208
EMBERTON, PEARCE & RANDALANA
TABLE 3. Analysis of variance in the number of species collected per 20 m x 20 m plot, with
least-squares estimates of means. Independent variables are sampling method (timed search vs. litter
sample vs. soil-plus-litter sample), elevation (200 m vs. 300 m), and location (one of three mountains).
Sum of Degrees of Mean Probability
Source Squares Freedom Square F-Ratio of Equality
Sampling 172.0 2 86.0 16.83 0.000***
Elevation 29.6 1 29.6 5.80 0.021*
Locality 28.3 2 10.2 1.99 0152
Sam x Elv 16.1 2 8.1 1.58 0.220
Sam x Loc 38.3 4 9.6 1.88 0.136
Elv x Loc 0.9 2 0.5 0.09 0.914
ХЕХЕ 10.6 4 PT 0.52 0.722
Error 184.0 36 51
Number of Species
Mean Std. Error N
Sampling:
Timed 9.0 0:5 18
Litter 4.7 0.5 18
Soil-Lit 6.3 0:5 18
Elevation:
200m 7.4 0.4 27.
300m 5.9 0.4 27
Locality:
Mahermano 5.9 0:5 18
llapiry 7.4 0.5 18
Vasiha 6.6 0.5 18
*p < 0.05, *** p < 0.001.
was partitioned out (see Emberton et al., in
review, concerning elevational variation).
Timed searching within 20 m x 20 m for three
person-hours averaged 9.0 species. This was
about twice as many species as occurred in
an eight-liter sample of litter selected from
the same area (4.7 species), and was about
half again as many species as occurred in an
equivalent soil-plus-litter sample (6.3 spe-
cies). Thus, this timed searching method pro-
duced more species than the other two sam-
pling methods. When considered in the
context of time invested, the productivity of
timed searching by this method was even
more pronounced (see above).
Timed searching alone, however, fell far
short of assessing total number of species
collected. ANOVA results in Table 4 indicate
that timed searching produced on average
only 72% of the species sampled within a 20
m x 20 m plot. Thus, the number of species
found in a plot during three person-hours of
searching was boosted 39% (28%/72%) by
one eight-| sample each of litter and soil-
plus-litter. Most of these additional species
occurred in soil-plus-litter samples, which
yielded half of the total, as opposed to the
litter samples, which yielded only somewhat
over a third of the total sampled species.
On the other hand, Table 5 shows that for
sampling live-collected individuals, litter
sampling was equivalent to timed searching
(51.6 +5.6 = 46.4 +5.2) and significantly more
efficient than soil-plus-litter sampling. Thus,
nearly half of the litter-sample and timed-
search species were represented by at least
one live-collected individual, whereas only
somewhat over a fourth of the soil-plus-litter-
sample species were. This result was not sur-
prising because soil can accumulate dead
shells of snails living in litter or trees (pers.
observ.; Burch 4 Pearce, 1990). In other
words, litter sampling and timed searching
both yielded more than 1.5 times the propor-
tion of live-collected species as soil-plus-lit-
ter sampling.
Table 6 shows the total live-plus-dead
number of each species collected by each of
the three sampling methods. Twenty species
(25%) were represented by at least 1% (> 24)
of the total specimens. Chi-square tests on
these species indicated that 12 (60%) of
them had equal (not significantly different)
representation among sampling methods. Of
RAINFOREST LAND-SNAIL SAMPLING 209
TABLE 4. Analysis of variance in the percent of species that were collected within each replicate plot,
with least-squares estimates of means. the independent variable is sampling method (timed search vs.
litter sample vs. soil-plus-litter sample).
Sum of Degrees of Mean Probability
Source Squares Freedom Square F-Ratio of Equality
Sampling 30,530.9 2 15,265.4 49.1 0.000***
Error 43,868.3 141 Salen
Percent of Species
Mean Std. Error N
Sampling:
Timed 72.4% 2.5% 48
Litter 37.1% 2.5% 48
Soil-Lit 50.1% 2.5% 48
*#* р << 0.001.
TABLE 5. Analysis of variance in the percent of species represented by at least one live-collected
individual, with least-squares estimates of means. Independent variables are sampling method (timed
search vs. litter sample vs. soil-plus-litter sample), elevation (200 m vs. 300 m), and location (one of
three mountains).
Sum of Degrees of Mean Probability
Source Squares Freedom Square F-Ratio of Equality
Sampling 5057.0 2 2528.5 5.24 0.010
Elevation 165.1 1 165.1 0.34 0.562
Locality 922.9 2 461.5 0.96 0.394
Sam x Elv 381.0 2 190.5 0.40 0.677
Sam x Loc 1446.5 4 361.6 0575 0.565
Elv x Loc 712.9 2 356.4 0.74 0.485
SES 2627.2 4 656.8 1.36 0.268
Error 16405.0 34 482.5
Percent Live Species
Mean Std. Error N
Sampling:
Timed 46.4% 5.2% 18
Litter 51.6% 5.6% 16
Soil-Lit 28.4% 5.2% 18
Elevation:
200m 43.9% 4.5% 25
300m 40.3% 4.2% 27
Locality:
Mahermano 47.8% 5.4% 17
llapiry 41.3% 5.2% 17
Vasiha 37.3% 5.4% 17
bp = 0:01:
the remaining eight species, Boucardicus sp.
9 and Microcystis sp. 4 were significantly
more prevalent in both litter and soil-plus-
litter samples than in timed-search samples,
and Sitala sp. 7 was present in the litter sam-
ples in greater proportions than expected in
the chi-square test. All three of these are both
dark brown in color (matching the color of
litter and soil) and minute in size (adult great-
est dimensions 2.2 mm, 2.2 mm, and 1.8
mm, respectively).
Two species—Streptaxidae spp. 9 and
13—were predominant in soil-plus-litter sam-
ples and notably scarce in litter only samples.
Both these species are high-spired (height/
diameters 2.5 and 2.2), very small (adult
210 EMBERTON, PEARCE & RANDALANA
TABLE 6. Numbers of snails of each of 80 species collected using three different sampling methods: t =
timed search, | = litter sample, s = soil-plus-litter sample. Chi-square tests for equal frequencies among
sampling methods were calculated for each species with > 24 specimens: * p < 0.05, Bonferroni
adjusted. GnSp = genus or family and numbered morphospecies. Genera and families in taxonomic
order are: BO, Boucardicus; CY, Cyathopoma; MN, Malarinia; TR, Tropidophora; OM, Omphalotropis;
FA, Fauxulus; SU, Subulinidae; ST, Streptaxidae; CH, Charopidae; KL, Kaliella; Ml, Microcystis; KD,
Kalidos; MG, Malagarion; and $1, Sitala.
Number
GnSp t | $ Total Chi-Sq
BOO1 IS 35 43 209 5.4
BOO2 47 11 15 ES 2.5
B003 3 0 2 5 —
BO04 11 5 8 24 —
BO05 2 0 1 3 —
BO06 6 3 0 9 —
BOO7 32 10 9 51 2.4
B008 2 0 3 5 —
BO09 1 39 33 73 102237
BO10 1 1 1 3 —
BO11 7. 0 5 12 --
BO12 1 0 1 2 =-
BO13 5 5 6 16 —
BO14 2 2 2 6 —
BO15 0 1 0 1 --
BO16 1 1 0 2 —
BO17 0 1 0 1 =
CY01 13 9 12 34 4.3
MNO1 0 0 1 1 —
TRO1 195 20 56 271 33:0:
OMO1 0 1 2 3 —
OMO2 1 Uf 1 9 -
FAO1 1 2 1 4 —
FAO2 0 0 1 1 -
SU01 104 18 54 176 6.3
SU02 3 2 2 7
SU03 2 5 6 13 —
STO1 9 0 1 10 —
STO2 6 1 3 10 —
STO3 11 2 0 13 —-
$104 9 3 8 20 —
STO5 2 0 0 2 —
ST06 88 25 53 166 2.0
STO7 20 2 if 29 2.9
STO8 us 1 4 18 —
$109 13 1 25 39 27.8
$110 2 11 7 20 —
Sidi 2 2 5 9 —
Sin2 6 1 3 10 —
Sii3 10 8 27 45 26.7
ST14 3 0 1 4 =
heights 3.9 mm and 3.6 mm), and with
glossy, fusiform, small-apertured shells sug-
gestive of a soil-burrowing niche. In contrast,
Tropidophora sp. 1, Kalidos sp. 1, and Sitala
sp. 5 all occurred predominantly in timed
searches and were significantly under-repre-
sented in litter and soil-plus-litter samples. All
Number
GnSp t | $ Total Chi-Sq
CHO1 28 4 3 35 9.0
CHO2 75 34 59 168 8.2
CHO3 6 0 3 9 —
CHO04 14 1 12 27 5.9
CHO5 14 2 5 21 —
CHO6 18 2 8 28 PA
CHO7 3 0 0 3 —
CHO8 1 0 0 1 —
CHO9 4 0 0 4 —
KLO1 8 6 1 15 —
М1 15 3 4 22 —
MIO2 7 0 1 8 —-
MIO3 Si 9 14 54 0.1
MIO4 1 9 20 30 34.6*
MIO5 1 0 2 3 —
MIO6 2 1 0 3 —
MIO7 0 1 0 1 —
MIO8 2 4 2 8 —
м9 3 2 0 5 —
MI10 2 0 1 3 —
MI11 1 0 0 1 —
MI12 1 0 0 1
KDO1 105 9 21 135 Pia
KDO2 22 2 0 24
KDO3 it 1 0 8 =
KDO4 6 4 0 10 —
KDO5 1 0 0 1 —
KDO6 И 1 1 9 a
KDO7 9 11 1 21 —
MGO1 9 3 1 13 —
$101 11 5 3 19 —
$102 т 0 3 10 —-
$103 1 1 4 6 —-
$104 5 1 3 9 —-
$105 49 0 2 51 34.2*
$106 4 0 0 4 —
$107 102 70 78 250 27.8*
$108 1 0 0 1 —
$109 0 0 1 1 —
Tot 1348 420 662 2430
three of these species are relatively large
(adult greatest dimensions 13.1 mm, 33.5
mm, and 7.3 mm). Tropidophora sp. 1 is of-
ten if not exclusively arboreal, and K. sp. 1
juveniles are at least partially arboreal, as
they frequently show up in vegetation-beat-
ing samples (Emberton, unpublished); S. sp.
RAINFOREST LAND-SNAIL SAMPLING 211
5 has a fragile, light-colored shell that is high-
spired for the genus (height/diameter 1.0), all
suggestive of arboreality.
A total of 101 specimens passed through
the 1.2-mm sieve. (Distributions of these
specimens among species and samples are
archived at MBI and ANSP.) Thus, the
1.2-mm sieve caught a minimum of 78% of
the specimens in the litter and litter-plus-soil
samples of each plot.
The 1.2-mm sieve caught representatives
of all species in the samples, however, ex-
cept for one: Streptaxidae sp. 15. This is a
minute, high-spired species (adult height 2.4
mm, diameter 1.0 mm), of which only two
specimens were obtained. In addition, the
sieve passed at least one adult of the five
smallest species of Boucardicus, some in
substantial numbers. Thus, adults of six spe-
cies (8% of total) passed through the sieve at
least in part. The smallest adult dimension of
any of these species was 1.0 mm.
DISCUSSION
Sieving of litter and litter-plus-soil may at
first seem superior to timed searches for
sampling land-snail diversities because it
yields higher ratios of species to individuals.
Practically, however, timed searches are the
most expedient by far, yielding species at 6.6
times the rate per person hour of either
sieved sampling method. The degree of this
advantage is surely an overestimate, be-
cause of our labor-intensive method of wet-
sieving then picking for all invertebrates; nev-
ertheless, even if we could halve or quarter
our litter-processing time, time searching
would be 3.3 or 1.7 times as efficient. Timed
searching also requires minimal equipment
and minimal weight and volume of samples
to transport (critical factors in expeditions
that require extensive backpacking).
Nevertheless, our method of timed search-
ing yielded fewer than three-fourths of the
total species collected per plot. A more thor-
ough sampling strategy must, therefore, in-
clude some litter or soil-plus-litter sampling.
Both these methods were roughly equivalent
in their species richness and number of spec-
imens per person-hour of effort. There were
different advantages to each. Litter samples
were 50% faster to process and yielded more
live-represented species, whereas soil-plus-
litter samples collected burrowing species
that were otherwise missed. Thus a sample
of litter-plus-soil seems preferable.
Thus, for greatest efficiency in assessing
species number and obtaining live speci-
mens, a good strategy seems to be collect-
ing litter-plus-soil samples during timed
searches, taking them from places that are
yielding good numbers of micro-molluscs. To
be quantifiable, samples should be taken to a
constant or measurable volume.
Because only minute, cryptic or burrowing
species were missed by timed searching, be-
cause the 1.2-mm sieve passed both adults
and identifiable juveniles, and because 1.0
was the smallest adult dimension we en-
countered, we recommend in processing the
supplemental litter-plus-soil samples that the
5.5-mm sieve fractions be discarded, and
that both the > 1.2-mm and the > 0.85-mm
fractions be retained and picked for micro-
molluscs. Only a few of the minutest juveniles
will be missed, at least for these Madagascar
samples. Because wet-sieving is very labor-
intensive (Table 2) and logistically difficult, we
recommend dry-sieving, either on-site when
litter and soil are dry enough, or later when
the samples have been stored in, for exam-
ple, muslin bags long enough to dry suffi-
ciently without dehydrating slugs and semi-
slugs.
Macro-molluscs (young juveniles > 5 mm)
tend to comprise only a small part of the
Madagascan rain-forest land-snail fauna, in
this case 7% (6/88) of the species. Also,
macro-snails have been the most extensively
collected in the past (Emberton, 1995b), so
are least likely to yield new biogeographic or
systematic information. Therefore, for great-
est efficiency in sampling total species rich-
ness, we recommend emphasizing the col-
lection of micro-snails, and collecting macro-
snails only as they are encountered during
micro-snail searches.
Thus, in sum, timed searches for micro-
snails, incidentally collecting macro-snails
and litter-plus-soil for dry-sieving and picking
the > 1.2-mm and > 0.85-mm fractions, seem
best for quantitatively sampling Madagascan
rainforest land-snails. This strategy should
be transferable, with local modifications, to
other tropical rainforests.
ACKNOWLEDGMENTS
We are grateful to the U.S. National Sci-
ence Foundation and USAID for funding
(grant DEB-9201060 to KCE); to the Mada-
212 EMBERTON, PEARCE & RANDALANA
gascar Département des Eaux et Forets
(DEF) for permission to collect and export
specimens; to the staff of Ranomafana Na-
tional Park Project (RNPP) (especially P.
Wright, B. Andriamahaja, L. Robinson, and
Madame Aimée) for logistic support; to Rich-
ard laly, Edmond of Ambatolahy, and our
other collectors and guides from the villages
of Esetra, Mahialambo, and Malio for their
superb work and their stamina under trying
conditions; to Gervais of RNPP for his amaz-
ing feats of driving; to the officials and other
residents of Esetra, Mahialambo, and Malio
for their very generous logistic support; to our
sorting assistants in Fort Dauphin, Ranoma-
fana National Park, and Antananarivo for their
careful labor; to M. Fenn (World Wide Fund
for Nature, Fort Dauphin) for recommending
Malio and its forests; to the DEF chef, Fort
Dauphin, for recommending Esetra Forest;
and to C. Hesterman (MBI) for help with data
analysis.
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SEQUENCING METHODOLOGY AND PHYLOGENETIC ANALYSIS: CYTOCHROME
b GENE SEQUENCE REVEALS SIGNIFICANT DIVERSITY IN CHINESE
POPULATIONS OF ONCOMELANIA (GASTROPODA: POMATIOPSIDAE)
Christina M. Spolsky,' George M. Davis? 8 Zhang Yi?
ABSTRACT
The evolution of the snail species Oncomelania hupensis, a vector for transmission of Schis-
tosoma japonicum in Asia, is tightly linked to the evolution of its parasite. We report here on
studies of the evolution of O. hupensis on the mainland of China, using sequence divergence
of the mitochondrial cytochrome b gene. The cytochrome b gene was amplified by PCR, cloned
in pBluescript, and sequenced; these methods are described in detail. The sequences for three
populations of two subspecies of this prosobranch gastropod were aligned and used in re-
constructing phylogenetic trees. The phylogenetic analyses confirm the divergence of Chinese
Oncomelania into subspecies and provide a finer tool for further genetic discrimination. Com-
parison of Oncomelania cytochrome b sequences to published sequences for two pulmonate
gastropods shows a greatly increased divergence rate in the pulmonates relative to that of
Oncomelania and of other metazoan groups.
Key words: Oncomelania, sequencing methodology, phylogenetics, PCR, cloning, cy-
tochrome b, infraspecific diversity
INTRODUCTION
The rissoacean genus Oncomelania is of
particular importance to the field of tropical
medicine because one of its two species, the
polytypic Oncomelania hupensis, is involved
in the transmission of Schistosoma japoni-
cum in Asia.
Oncomelania minima is restricted to north-
western Honshu, Japan. Polytypic O. hupen-
sis, in contrast, is distributed from northern
Burma (fossil) throughout southern China,
Japan, the Philippines and Sulawesi. The
polytypic status of Oncomelania has been
reviewed (Davis, 1994) with the following
subspecies recognized: O. h. hupensis (China
mainland); O. h. formosana and O. В. chiui
(Taiwan); O. h. nosophora (Japan); O. h. qua-
drasi (Philippines); O. h. lindoensis (Sulawesi).
More recently, Davis et al. (1995) recognized
three subspecies on the mainland of China on
the basis of allozyme molecular genetics,
shell morphology and biogeography: O. h. hu-
pensis; O. В. robertsoni and O. В. tangi.
A coevolved relationship between snail lin-
eages and the genus Schistosoma extends
back to the Gondwanaland origin of these
taxa. Davis (1992), in reviewing the patterns
and processes of this coevolution, made the
point that transmission of the parasite now is
population-specific in many instances. The
hypothesis is that as populations of Oncomel-
ania have dispersed and diversified in the di-
rection from Burma-Yunnan, China, through-
out China to Japan and the Philippines (Davis,
1979), the parasite has had to modify genet-
ically with the genetically changing snail pop-
ulations or become regionally extinct. This hy-
pothesis predicts that genetic distances
among parasite populations parallel genetic
distances among snail populations.
Allozyme electrophoretic data demonstrate
strong population divergence among popu-
lations of Oncomelania throughout China
(Davis et al., 1995). The problem with the elec-
trophoretic approach, however, is that as one
increases the number of populations com-
pared, errors in assigning the homology of
alleles increase. Because one must always
run a control population as a standard for
determining the identity of alleles at each of 30
or more loci, one needs exponentially increas-
ing numbers of cross-comparisons among
populations; the experimental labor and need
to keep many fresh frozen populations be-
comes prohibitive.
The Academy of Natural Sciences of Philadelphia, 1900 Benjamin Franklin Parkway, Philadelphia, Pennsylvania, USA
19103
“Chinese National Center of Systematic Medical Malacology, Chinese Academy of Preventive Medicine, Shanghai, Peo-
ple’s Republic of China
214 SPOLSKY, DAVIS 4 YI
TABLE 1. Localities and collecting information for three populations of Oncomelania hupensis in China.
Latitudes and longitudes are given. Catalog numbers given are for the Chinese Institute of Parasitic
Diseases (CIPD) and the Academy of Natural Sciences of Philadelphia (ANSP).
1. Sichuan (SC):
Sichuan Province; TianQian County; Xing Hua District; Xia Len Village 2nd group.
102°46.0’E; 30°5.02’N; CIPD 0338
2. Yunnan (DA):
Yunnan Province; Dali City; Da Jin Ping Zi Ran Village. Ditch.
100°12.4’E; 25°27.6’N; CIPD 0349
3. JiangXi (JX):
JiangXi Province, Pengze County, WangLing District, JingWang Village.
116°30.0’E; 29°55.0’N; ANSP 399275
collected 7 Dec. 1993 by Zhang Jian Guo and Guo Gang Qiang
Gene sequencing provides an alternative
method for reconstructing and evaluating
phylogenetic relationships among a group of
organisms. Once a gene is sequenced, that
sequence is permanently available for com-
parisons, and does not need to be repeated
as taxa are added to the analysis. We have
looked for a gene that evolves rapidly enough
to distinguish populations of Oncomelania
to the same as or a greater degree than allo-
zymes do. We have not used RFLD (restric-
tion fragment length differences) methods
because they are too crude and because
they also rely on cross-comparisons of elec-
trophoretic mobility. We have not used mi-
crosatellites because they give too fine a res-
olution: they provide excellent discrimination
at the population level and below, but are too
sensitive a tool for inter-population and inter-
specific comparisons. Sequences from the
appropriate gene potentially provide the
most powerful set of discrete character data
for phylogenetic analysis.
This paper presents detailed methods and
preliminary results of amplifying and se-
quencing the mitochondrial cytochrome b
gene from Oncomelania hupensis, and com-
paring the results of a three-taxon analysis
with results from the allozyme study by Davis
et al. (1995). Problems encountered in PCR
amplification of cytochrome b from On-
comelania are stressed. Numerous system-
atic studies of cytochrome b gene sequence
document that the rate of evolution of this
gene is appropriate to demonstrate differ-
ences among species and infraspecific taxa.
Sufficient phylogenetically informative char-
acters are present in such comparisons so
that phylogenetic analyses are very robust. In
contrast to ribosomal RNA genes, there are
few or no insertions and deletions among cy-
tochrome b genes, and thus no problems
with alignment even among taxa as divergent
as molluscs, insects, and mammals. Be-
Cause correct alignment is essential to ob-
taining the correct phylogeny (Thorne & Kish-
ino, 1992), comparison of genes coding for
proteins allows one to reconstruct phyloge-
nies that are more likely to reflect true evolu-
tionary relationships for those genes.
MATERIALS AND METHODS
Specimens Studied
Snails were collected from three localities
in China: in Sichuan and Yunnan Provinces in
northwestern China, and in JiangXi Province
in the east (Table 1). Snails were brought to
the United States alive in an estivating state.
Once in the laboratory, snails were activated
by placing them on moist filter paper in Petri
dishes and kept at 4°C. Immediately prior to
isolation of DNA, the snails were quick-frozen
at —80°C by placing them individually in the
wells of a ceramic depression plate previ-
ously chilled to —80°C.
DNA Preparation
The methods used for preparing DNA from
individual snails were modified from those of
Spolsky & Uzzell (1984, 1986) and of Doyle &
Doyle (1987). Briefly, a frozen snail (4-9 mm
shell length) was crushed, the whole individ-
ual immediately dropped into 600 ul of lysis
buffer (0.02 M Tris, 0.1 M EDTA, 0.5% Sar-
kosyl) containing 200 ug/ml proteinase К,
and incubated at 55°C overnight. One hun-
dred ul of each of 5 M NaCl and CTAB ex-
traction solution (5% CTAB, 0.5 M NaCl)
were added, and the resulting solution ex-
tracted with an equal volume of chloroform.
800 ul of CTAB precipitation buffer (1%
CTAB, 50 mM Tris pH 8.0, 10 mM EDTA)
were added to the aqueous phase, mixed,
and placed at room temperature for 30 min.
The CTAB-DNA precipitate was pelleted (15
min at 14K rpm), redissolved in NTE (1.2M
CYTOCHROME b DIVERSITY IN CHINESE ONCOMELANIA 215
NaCl, 10 mM Tris, 1 mM EDTA) containing
100 ug/ml RNase, and again precipitated by
addition of two volumes of ethanol. The DNA
pellet was washed with 70% ethanol in TE,
then redissolved in 100-200 ul of water or
0.1x TE. Aliquots (3 ul) of each DNA were
subjected to electrophoresis through a 0.8%
agarose gel in TBE and stained with ethidium
bromide to obtain a rough estimate of the
DNA concentration and quality. The amount
of DNA was more precisely quantified using a
Hoefer TKO100 fluorometer. Concentration
of each DNA preparation was adjusted to 50-
100 ng/ul. Using this protocol, we obtained
between 5 and 65 ug of high molecular
weight DNA per individual snail.
DNA Amplification
PCR was used to amplify the mitochondrial
cytochrome b gene using the primer pair
On5L (forward: 5’-CATTTAGGTCTGCGGTC-
CAC) and On6H (reverse: 5’-GGCGTAAC-
TAGTGGGTTAGCTGG). These Oncomela-
nia-specific primers define a fragment 610 bp
in length. Preliminary sequence for O. hupen-
sis from Sichuan was obtained using mollus-
can primers SUP1 and SUP2 (a gift from T.
Collins). These primers, although not optimal,
provided sufficient sequence to enable de-
sign of On5L and On6H. Optimal sequence
for the latter primers was determined using
PRIMER version 0.5 (Lincoln et al., 1991) and
was based on the preliminary Oncomelania
sequence in combination with comparisons
of conserved cytochrome b regions for a
number of molluscan, echinoderm, and ver-
tebrate taxa. Each PCR reaction contained
approximately 50-100 ng of template DNA,
200 uM of each dNTP, 30 pmole of each
primer, and two units of Taq polymerase
(Promega), in 50 ul of supplier-provided
buffer at a magnesium concentration of 2.5
mM. The PCR conditions consisted of 40 cy-
cles of denaturation at 94°C for 45 sec, an-
nealing at 43°C for 1 min, and extension at
72°C for 1 min 20 sec on an M-J Research
model PTC-100 thermal controller.
Cloning, Screening and Sequencing
Amplified DNA products were separated
on a 1% agarose gel. Bands corresponding
to fragments of the correct size were cut out,
purified using Geneclean (Bio 101) glass
beads, and quantified by fluorometry. A
90-ng aliquot of the purified PCR product
was used for ligation into the polycloning re-
gion of the plasmid vector pBluescript SK
(Stratagene), previously prepared for ligation
by linearizing with EcoRV and ddT-tailing us-
ing a modification of Holton & Graham’s
(1991) method. For the latter protocol, the lin-
earized vector was incubated with ddTTP
and terminal transferase at 37°C for one
hour; 20 ng of this ddT-tailed vector was
used per ligation. Each ligation reaction con-
tained, in addition to PCR product and pre-
pared vector, 4% polyethylene glycol 8000
and 0.2-0.5 unit of T4 DNA ligase (Promega)
in the appropriate buffer. Ligations were al-
lowed to proceed overnight at 15°C, then
drop-dialyzed by placing each reaction on a
Millipore type VS25 membrane floating in a
Petri dish on 0.1x TE. One third of a ligation
was used for transformation via electropora-
tion (BioRad pulser) of the E. coli host cell line
XL1Blue. Bacterial colonies with recombinant
plasmids were identified by plating on selec-
tive Luria agar plates containing 100 mg/ml
ampicillin, 40 ug/ml X-gal, and 40 ug/ml
IPTG. Putative positive colonies were grown
overnight at 37°C in 2 ml of LB + ampicillin.
Minipreps (Sambrook et al., 1989) of these
growths were screened for the presence of
inserts of the correct size by cutting the insert
out of the recombinant plasmid with Hindlll
and Pstl, followed by electrophoretic analy-
sis. Confirmed positive colonies were grown
in larger scale liquid cultures (15 ml), and
plasmid isolated from them on Qiagen tip-
100 columns following the manufacturer’s
protocol. Sequences of the cloned fragments
were determined by automated cycle se-
quencing on an ABI 373A sequencer with
Stretch upgrade, using commercially avail-
able vector primers T3 and T7. Using the au-
tomated sequencer, these two primers pro-
vide completely overlapping sequence for
each strand of the 610 bp cytochrome b frag-
ment. To prevent incorrect nucleotide calls
caused by occasional random misincorpora-
tion of nucleotides during amplification, at
least three clones of each ligation were se-
quenced.
Data Analyses
Sequences for each individual were as-
sembled by visual inspection using the se-
quence editor ESEE version 1.09e (Cabot &
Beckenbach, 1989). ESEE was also used to
align Oncomelania sequences with each
other and with cytochrome b sequences
216 SPOLSKY, DAVIS 4 YI
available from Genbank. Aligned sequences
were formatted appropriately for phyloge-
netic analyses using EAT (Cabot, 1993). Pair-
wise maximum-likelihood distances were
calculated using program DNADIST of the
phylogenetic analysis package PHYLIP ver-
sion 3.57 (Felsenstein, 1989, 1993); these
distances were estimated under the Felsen-
sten maximum-likelihood model, which
takes into consideration unequal frequencies
of nucleotides, unequal rates of transitions
and transversions, and multiple substitutions
at individual sites. Distance, parsimony, and
maximum-likelihood trees were calculated
using programs FITCH, DNAPENNY, and
DNAML of PHYLIP, programs that do not as-
sume equal rates of change along the
branches of a tree. Optimal FITCH and
DNAML trees were found by running 20 rep-
etitions of each program with randomized in-
put order and optimization by global branch
rearrangement. Bootstrap and delete-half-
jackknife estimates (1,000 replicates) of con-
fidence intervals for the maximum-likelihood
analyses were made using program SEQ-
BOOT, in conjunction with DNAML and CON-
SENSE.
RESULTS
Relationships Among the Populations of
Oncomelania hupensis
Figure 1 presents aligned cytochrome b
sequences for individual O. hupensis from
three populations in China (Sichuan, Yunnan,
and JiangXi); each sequence represents the
consensus from at least three clones for that
individual. Sequences were obtained for two
individuals from the locality in Sichuan; diver-
gence between the two specimens was less
than 0.4% (two site differences). This low in-
trapopulation divergence is consistent with
low intrapopulational variability in morphol-
ogy as well as in other genetic measures.
Given the low intrapopulation relative to in-
terpopulation variability, we concentrated in
this preliminary study on obtaining measures
of cytochrome b divergence among, rather
than within, populations. Oncomelania se-
quences were also aligned with published
sequences for the gastropods Albinaria co-
erulea and Cepaea nemoralis. Nucleotides
corresponding to the primer regions have
been trimmed from the sequences, resulting
in alignment of 572 nucleotides. In this region
of cytochrome b, Cepaea and Albinaria share
an extra nucleotide triplet at positions 109-
111; in addition, Cepaea alone has an inser-
tion of two nucleotides at positions 521-522.
For the phylogenetic analyses, all nucleotide
positions were included. The three O. hupen-
sis sequences have relatively few changes
among them; a total of 70 variable sites were
detected in this region of the cytochrome b.
In contrast, A/binaria and Cepaea each differs
from Oncomelania at numerous nucleotide
positions. Sequence divergence estimates
are given in Table 2. Divergences between
the closest pair, Sichuan and Yunnan, are
3.8%, whereas between either of these and
JiangXi, distances are 10.2 and 11.9% re-
spectively. Distances between Oncomelania
and Albinaria average 53%, between On-
comelania and Cepaea, 58.8%.
Phylogenetic Analyses
For the phylogenetic analyses, the three
Oncomelania hupensis populations were
compared to АБтапа coerulea, Cepaea ne-
moralis, the sea urchin Strongylocentrotus
purpuratus, and the mammal Homo sapiens.
The stylommatophoran pulmonate gastro-
pods Albinaria and Cepaea are the closest
relatives to Oncomelania for which published
cytochrome b sequence is available. Strongy-
locentrotus and Homo were included in the
analyses to provide rooting for the gastropod
clade. The optimal transition/transversion ra-
tio, that is, the ratio which minimizes the like-
lihood measure, was determined empirically
using DNAML (P. Beerli, pers. comm.); for the
set of taxa used, the optimal ratio was 1.1. For
comparison, phylogenies were also recon-
structed using distances and parsimony. The
topology of the phylogenetic tree obtained by
each of the three methods was the same (Fig.
2). The validity of each tree was tested by both
bootstrap and jackknife resampling of the
data. For both resampling methods with all
tree building strategies, each node of the tree
was strongly supported by high bootstrap val-
ues (minimum of 95% confidence level).
DISCUSSION
Although cytochrome b sequencing has
been used in numerous systematic studies in
organisms ranging from vertebrates to ar-
thropods to echinoderms, it was not a simple
matter to apply those techniques to mol-
luscs. Because of the very ancient branching
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
Sichuan
Yunnan
JiangXi
ALBINARIA
CEPAEA
CYTOCHROME b DIVERSITY IN CHINESE ONCOMELANIA
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Tess:
FIG. 1. Alignment of sequences of a 572 nucleotide fragment of cytochrome b from three populations of
Oncomelania hupensis in China and from two pulmonate gastropods. The top line contains sequence for
cytochrome b of Oncomelania hupensis from Sichuan Province, China. For the remaining sequences (O.
hupensis from Yunnan and JiangXi Provinces, Albinaria coerulea, Cepaea nemoralis), nucleotides were
given only for sites that differed from the Sichuan sequence. A dot (.) indicates the presence of the same
nucleotide as in the top sequence; a dash (-) indicates the absence of a nucleotide at that position.
Sequences have been deposited with Genbank.
of molluscs from the basal phylogeny, their
cytochrome b sequences are divergent
enough so that the so-called “universal prim-
ers” for cytochrome b (Kocher et al., 1989)
do not amplify this gene from molluscs. In
fact, there is enough divergence within the
molluscs to make designing a universal
primer for Mollusca difficult. The PCR prim-
ers we designed, On5L and On6H, work for
most, but not all, populations of Chinese On-
comelania, and variably well for the related
Tricula. Because of the variable yield and pu-
rity of the product from different taxa, we
cloned all amplified products prior to se-
quencing. Although more labor-intensive,
this procedure provided such excellent qual-
ity sequence that in the long run time was
saved by not having to do multiple repetitions
218
SPOLSKY, DAVIS 4 YI
Strongylocentrotus
97
y Yunnan
ANA
100 Sichuan
Homo
JiangXi
AAA Cepaea
a А
Albinaria
FIG. 2. А maximum-likelihood tree of relationships among three populations of the mesogastropod proso-
branch Oncomelania hupensis and the stylommatophoran pulmonates Albinaria coerulea and Cepaea
nemoralis. Both Homo sapiens and Strongylocentrotus purpuratus were used as outgroups for the analysis
in order to provide rooting for the gastropod node and allow for calculation of a bootstrapping value for the
node. Bootstrap values are listed to the left of each node; these indicate the number of times that node
occurred among 1,000 bootstrap replicates.
and by the clarity and thus accuracy of the
sequence obtained.
The three populations of Oncomelania hu-
pensis form, as expected from divergence
levels, a very closely related monophyletic
group. Previous conchological, biogeo-
graphic, and electrophoretic analyses (Davis
et al., 1995) suppont division of O. hupensis
into three subspecies: the Sichuan and Yun-
nan populations belong to the subspecies O.
h. robertsoni, whereas the JiangXi population
belongs to the subspecies O. h. hupensis.
The phylogenetic analyses of nucleotide se-
quence of cytochrome b presented here are
consistent with this subspecies concept: the
Sichuan and Yunnan populations are the
most closely related (Fig. 2, Table 2). These
populations are geographically close, and
have smooth shells with no varix. Distances
of either of these populations to the JiangXi
population, ribbed and with a strong varix,
are almost threefold more.
The two pulmonate gastropods, Albinaria
and Cepaea, form a separate group that di-
verged over 300 million years ago from the
prosobranch Oncomelania. Both gastropod
groups, Oncomelania and pulmonates, share
a common node relative to non-molluscan
taxa. One striking feature of the phylogenetic
tree is the very long branch lengths for the
pulmonates, particularly for Cepaea (Fig. 2),
suggesting many more changes along the
branches leading to Albinaria and Cepaea
than along any other branches. This is also
evident in pairwise distance comparisons
(Table 2). In comparisons using either Ce-
paea or Albinaria, results are what one would
expect: distances to Oncomelania are less
than to Homo or Strongylocentrotus; on the
other hand, using Oncomelania, pairwise dis-
tances to Homo and to Strongylocentrotus
are less than to the more closely related gas-
tropod taxon Cepaea. This is not a phenom-
enon restricted to the cytochrome b gene:
phylogenetic analyses of cytochrome oxi-
dase (Hoeh et al., 1996) have also demon-
strated a longer branch length for Albinaria.
In agreement with the large divergences we
find for the pulmonates, the mitochondrial
gene order for these two taxa also appears to
have changed extensively, both from that of
other molluscs as well as from other meta-
zoan groups (Lecanidou et al., 1994; Hatzo-
glou et al., 1995). An increased rate of evo-
lution of the mitochondrial genome and of its
gene order has also been observed in the
bivalve mollusc Mytlius edulis (Hoffmann et
al., 1992; Hoeh et al., 1996), but not in the
polyplacophoran mollusc Katharina tunicata
(Boore & Brown, 1994).
The increased cytochrome b divergence
in pulmonates is puzzling. Some of the extra
length of the pulmonate branches may pos-
sibly be a result of frameshift-causing mis-
readings of the nucleotide sequence. For
example, if we translate the nucleotide se-
CYTOCHROME b DIVERSITY IN CHINESE ONCOMELANIA 219
TABLE 2. Pairwise comparisons of sequence divergence over 572 nucleotide positions in the
cytochrome b gene. Sequence divergences were estimated using the program DNADIST of PHYLIP
version 3.57, under Felsenstein’s (1989) maximum likelihood method.
S У JX
Sichuan —
Yunnan 0.0382 —
JiangXi 0.1021 0.1186 —
CEPAEA 0.5969 0.5853 0.5899
ALBINARIA 0.5364 0.5427 0.5256
HOMO 0.5158 0.5032 0.5067
STRONGYLO 0.5409 0.5304 0.5324
quence of Cepaea downstream from the
point of the two-nucleotide insertion (Fig. 1),
and compare it to the Sichuan Oncomelania
sequence, we find virtually no homology
among the 14 amino acids coded (Fig. 3);
however, if we delete these two nucleotides
from the Cepaea sequence, homology of the
translated sequences becomes significantly
higher. Possible sequence misreadings re-
sulting in sequential insertions and deletions
relative to other sequences may be difficult to
detect if the sequence eventually goes back
into alignment. If such shifts are not allowed
for, however, this would result in significantly
longer pairwise distances between taxa. Al-
ternatively, if the apparent frameshift muta-
tions reflect the real sequence rather than
misreadings of the sequence, then the mito-
chondrial cytochrome b sequences in these
two cases appear to behave as if they were
nuclear pseudogenes (cf. Collura 8 Stewart,
1995). Because these sequences were ob-
tained from cloned mitochondrial genomes, it
is unlikely that these do represent nuclear
pseudogenes. In this case, the apparent in-
creased rate of evolution of the mitochondrial
cytochrome b in the pulmonates suggests a
decrease in functional constraints for this
protein.
Some workers have avoided the apparent
high labor costs of gene sequencing by using
RFLD' analyses instead. This technique,
however, has many of the same problems as
“Although the abbreviation RFLP is often used to refer to
restriction fragment analysis of sequence variation, we
consider this inappropriate usage of the term polymor-
phism as applied to genetic analysis. Genetic polymor-
phism has a very specific meaning (cf. Ford, 1965) involv-
ing discontinuous variation, that is, distinct alleles, of
specific genes (genetic loci). In the restriction fragment
analysis method commonly termed RFLP, fragment mo-
bility differences cannot be assigned to specific loci. We
therefore intentionally use the term RFLD (restriction frag-
ment length difference) for this method.
CEP ALB HOMO STR
0.5148 =
0.7180 0.6190 =
07223 0.6177 0.4663 =
does genetic analysis of allozyme mobility
differences, and it often provides even less
information than allozyme analysis does. Two
additional serious weaknesses of RFLD anal-
ysis, not present in allozyme studies, are: (1)
the inability to identify genetic loci and there-
fore to know what is allelic to what; because
of this, one does not know which characters
are independent, a requirement for phyloge-
netic analysis. One therefore can at best get
only a very crude measure of similarity; (2) the
paucity of characters on which to base a phy-
logenetic analysis. A valid analysis requires a
larger number of variable characters than the
number of taxa; this is not the case for many
RFLD studies. These weaknesses probably
account for the discrepancy between the
conclusions of Hope & McManus (1995)
based on RFLD analyses and our conclu-
sions based on phylogenetic analyses of
cytochrome b gene sequence: among popu-
lations from a number of Oncomelania sub-
species from China, the Philippines, and Ja-
pan, the largest difference Hope & McManus
(1995) found is between the Sichuan and
Yunnan populations of Chinese Oncomela-
nia. Not only is this at variance with our elec-
trophoretic and sequencing results, but it is
also in conflict with conchological and bio-
geographic data. Our conclusions, on the
other hand, are strongly supported: there is
strong correspondance between our se-
quence data and biogeographic, concholog-
ical, and extensive allozyme data.
Ribosomal RNA sequencing has been
used extensively in determining phylogenetic
status among molluscs. In Oncomelania, se-
quences from the D6 domain and the 5’ ter-
minus of 23S-like rRNA were useful in deter-
mining the phylogenetic relationship of the
genus to a number of molluscan taxa, but
were not helpful in resolving finer differences
at the species level (Emberton et al., 1990;
Rosenberg et al., 1994). The present report
220 SPOLSKY, DAVIS & YI
Sichuan LVLFAPOMLTDPENFI
CEPAEA LLCYITL.YLRTPKTF
CEPAEA-2 VENEN SPS У
FIG. 3. Alignment of translated sequences for а
portion of the cytochrome b gene 3’ from position
523 of Fig. 1. The invertebrate genetic code was
used for translation. Amino acids were given only
for sites that differed from the Sichuan amino acid
sequence; a dot indicates the presence of the
same amino acid as in the Sichuan sequence. Ce-
paea = amino acid sequence translated from nu-
cleotide sequence stored in Genbank, accession
U23045; Cepaea-2 = two “extra” nucleotides, at
positions 521 and 522 of the amplified cytochrome
b sequence, were removed prior to translation.
confirms the utility of nucleotide sequencing
of cytochrome b; this gene provides the res-
olution necessary to determine intraspecific
relationships among populations of On-
comelania, and to cluster the populations ac-
cording to subspecies status. Work is in
progress, using cytochrome b gene se-
quencing, on the degree and patterns of ge-
netic divergence among Oncomelania hu-
pensis, other species of Oncomelania, and
Tricula. This work will serve to establish pat-
terns of divergence, to substantiate the sub-
species concepts for Oncomelania in China,
to place the differentiation of Oncomelania in
a phylogeographic context, and to corrobo-
rate the existence of two diverging subfami-
lies in the pomatiopsid assemblage, that is,
the Pomatiopsinae (Oncomelania) and Tricu-
linae (Tricula).
ACKNOWLEDGEMENTS
This work was supported in part by N.I.H.
grant TMP Al 11373 to Davis and, in part, by
а TMRC grant 1 P50 AI39461-01. The sup-
port of the Institute of Parasitic Diseases,
Chinese Academy of Preventive Medicine, is
gratefully acknowledged. We thank Thomas
Uzzell and two anonymous reviewers for crit-
ical comments on the manuscript.
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MALACOLOGIA, 1996, 38(1-2): 223-227
LEMERS:10 THE EDITOR
CRITERIA FOR THE DETERMINATION OF TAXONOMIC BOUNDARIES
IN FRESHWATER UNIONOIDS (BIVALVIA: UNIONOIDA):
COMMENTS ON STIVEN AND ALDERMAN (1992)
Walter В. Hoeh' & Mark E. Gordon”
The southern Atlantic Slope region of the
United States is an area characterized by high
species richness and considerable local en-
demism in the freshwater fauna (e.g., fresh-
water mussels [Johnson, 1970; Burch, 1975;
Davis et al., 1981; Kat, 1983; Hoeh, 1990],
snails [Burch 4 Tottenham, 1980; Thompson,
1968, 1984], crayfish [Hobbs, 1989], and fish
[Lee et al., 1980]). Kat (1983) demonstrated
species-level divergence within the regional
Lampsilis radiata (Gmelin 1791) complex (i.e.,
L. radiata s.s., L. sp. [now L. fullerkati Johnson
1984], and L. splendida [Lea, 1838]). Using
allozymic and morphological comparisons,
Stiven 4 Alderman (1992; hereafter referred to
as SA) examined nine populations of union-
oids from North Carolina in order to, among
other objectives, reassess and resolve the
taxonomic status of three nominal taxa of
Lampsilis: L. radiata radiata, L. radiata con-
spicua (Lea 1872), and L. fullerkati (taxa as
listed by Turgeon et al., 1988). SA concluded,
based primarily on genetic distance criteria,
that the former two taxa, and probably L.
fullerkati as well, should ‘be considered sim-
ply as allopatric ‘populations’ of Lampsilis ra-
diata.”
A critical reading of SA reveals inconsis-
tencies and errors in methodology and data
interpretation. We believe that the following
detailed discussion of SA is necessary for
two reasons: (1) The taxonomic revisions
suggested by SA, if implemented, would
likely have a significant impact on the level of
legislative protection afforded at least two of
the taxa in question, and (2) the high visibility
of this paper among malacologists and envi-
ronmental resource professionals may lead
to its use as an exemplar for studies of pop-
ulation structure, taxonomic boundaries, and
phylogenetic relationships in unionoid bi-
valves. Thus, because of the potential impact
of this paper, aspects of the research pre-
sented therein should be carefully re-evalu-
ated.
Morphological Analyses
The data presented in SA (table 2) suggest
that there are statistically significant differ-
ences in length, height, and width among
Lampsilis r. conspicua, L. r. radiata, and L.
fullerkati shells. However, similarity in slope
and y-axis intercept for plots of length versus
height for L. r. conspicua and L. r. radiata
(specimens of L. fullerkati not plotted) was
used as an indicator of conspecificity in SA
(fig. 2). Furthermore, the statistically signifi-
cant mensurable differences among the
above three Lampsilis taxa were downplayed
by reference to “site effects” in Lampsilis
cariosa and Leptodea ochracea (SA: 366).
Appeals to site effects (phenotypic plastic-
ity) as explanations for the observed concho-
logical differences among unionoid popula-
tions, without the appropriate substantiating
data, are simply hypotheses to be tested.
Contrary to statements in the text, the Deep
River Lampsilis cariosa population is not al-
lozymically identical to the other two popula-
tions of L. cariosa (SA: table 3). Therefore, the
data presented in SA cannot discount ge-
netic effects for the size differences among
populations of L. cariosa. Furthermore, be-
cause nine of the alleles detected in Lep-
todea ochracea were found only in one of the
two populations analyzed (again contrary to
the text; see SA: table 3), genetic effects can-
"Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada. Corresponding Author & Current
Address: Walter R. Hoeh, Department of Zoology, Miami University, Oxford, Ohio 45056, U.S.A.
“Zoology Section, Campus Box 315, University of Colorado Museum, Boulder, Colorado 80309, U.S.A.
224 HOEH & GORDON
not be ruled out in this instance either. Even
if two populations of a single species are al-
lozymically identical, distinct genetic deter-
minants for conchological morphology may
be present.
We wonder if the “site effect analyses”
presented in SA controlled for age and/or
gender (sexual dimorphism). These poten-
tially confounding factors were not discussed
in SA (no explanation of the methodology uti-
lized in the conchological analyses was pre-
sented in the “Methods” section). For exam-
ple, assuming no genetic component for the
observed size variation, were the Deep River
individuals of Lampsilis cariosa larger be-
cause of site effects or because they were
older? The Deep River “. . . population is
comprised of only large old specimens and is
thought to be declining” (SA: p. 356). We be-
lieve this statement to be suggestive of an
age effect that could have confounded the
conchological analyses. In summary, the sig-
nificant mensurable differences between L. r.
conspicua, L. r. radiata, and L. fullerkati, com-
bined with no substantiation for site effects,
are consistent with the hypothesis that these
three taxa are distinct evolutionary lineages.
The similarity in slope and y-axis intercept for
plots of length versus height for L. r. con-
spicua and L. r. radiata is irrelevant given the
significant morphological differences be-
tween these taxa.
Sampling Strategy for the Allozyme Study
“We note also that when distance mea-
sures are relatively large between pairs of
species, and heterozygosity is low, the con-
struction of phenograms can be carried out
fairly reliably with only a few representative
individuals for a species (Nei, 1978)” (SA:
357-358). However, the particular sampling
strategies used in SA for organisms and loci
will often produce unreliable estimates of ge-
netic distances. Three problems are outlined
below.
Although only single populations of Lamp-
silis r. conspicua and L. r. radiata were ex-
amined allozymical'y in SA, it is desirable to
use multiple populations to represent each
taxon in analyses of taxonomic boundaries
and among-taxa relationships (e.g., Baver-
stock & Moritz, 1990). This is especially true
for groups, such as the Unionoida, that are
known to contain cryptic species (e.g., Davis
et al., 1981; Davis, 1983, 1984). Furthermore,
representing species by single populations
can produce phylogenetically misleading re-
sults (e.g., Smouse et al., 1991).
Species of Drosophila are typically consid-
ered to have “high” heterozygosities (15-
20%; e.g., Gorman & Renzi, 1979). Therefore,
the heterozygosities reported in SA (table 3)
for the nine unionoid populations are not gen-
erally “low” (range: 5.1% to 31.8%, mean =
18.3%). Thus, estimates of genetic distances
reported in SA may be inaccurate due to the
small number of individuals used in certain
comparisons (e.g., LAP was assayed for a
single individual of Lampsilis fullerkati [SA: ta-
ble 3]).
Nei (1978) makes it clear that a relatively
large number of assayed loci are required for
accurate estimates of genetic distance. Nei
(1978: 583) stated the following: “Ме! and
Roychoudhurry (1974) concluded that for es-
timating average heterozygosity and genetic
distance a large number of loci rather than a
large number of individuals per locus should
be used. ...’ What quantity does Nei imply
with the phrase, “a large number of loci”? “In
fact, less than 30 loci were studied in most
recent protein surveys. This number is small;
ideally, more than 50 loci should be used... .”
(Nei, 1978: 587). The import of a relatively
large number of loci for estimation of genetic
distances has been empirically substantiated
(e.g., Gorman 4 Renzi, 1979). Both Nei’s and
Roger's distances are subject to large stan-
dard errors especially at relatively small dis-
tances (as is potentially the case among
Lampsilis fullerkati and the two populations
representing L. r. conspicua and L. r. radiata
analyzed in SA), and the major factor influ-
encing the standard errors is the number of
loci sampled (e.g., Nei, 1978, 1987; Nei 8
Chesser, 1983; Richardson et al., 1986;
Chakraborty & Leimar, 1987; Baverstock &
Moritz, 1990). The sampling of eleven loci, as
was the case in SA, does not give rigorous
estimates of genetic distance. Therefore, the
estimates of absolute genetic divergence pre-
sented in SA should be considered tentative
as should any taxonomic revision based on
those estimates.
Lack of Reference to Types
Neither Lampsilis r. radiata nor L. r. con-
spicua from their respective type localities or
type locality drainages (Potomac and Yadkin
TAXONOMIC BOUNDARIES IN FRESHWATER UNIONOIDS 225
rivers, respectively) were allozymically ana-
lyzed in SA. Not utilizing topotypic or near-
topotypic material for molecular evaluation
compounded with the lack of reference to
type material of any sort should preclude the
taxonomic revisions suggested in SA. Taxo-
nomic revisions must be based on reference
to “types.” Molecular analyses do not obvi-
ate this necessity. As it now stands, the
specimens of L. r. radiata and L. r. conspicua
utilized in SA cannot be confirmed as actually
representing the taxa implied.
Taxonomic Concepts
Although often referring to conchological
data, SA displays a strong reliance on levels
of genetic distance for the determination of
taxonomic boundaries. However, cogent ar-
guments, based on theoretical and opera-
tional criteria, have been made against the
use of genetic distances for delimiting taxa
(e.g., Frost 8 Hillis, 1990). A major operational
problem discussed in Frost & Hillis (1990) is
the arbitrary nature of genetic distance mea-
sures. Because allozyme loci evolve at differ-
ent rates (e.g., Sarich, 1977; Skibinski &
Ward, 1982), genetic distance estimates are
sensitive to the particular loci analyzed. Even
if identical loci are scored, distance estimates
may differ dramatically from one analysis to
another due to the use of different electro-
phoretic conditions (e.g., Singh et al., 1976).
The use of non-identical suites of allozyme
loci combined with different electrophoretic
conditions for some of the loci in common
between studies may partly explain the dis-
crepancy in reported genetic distance esti-
mates between Lampsilis fullerkati and L. ra-
diata radiata (SA: Nei’s D = 0.049; Kat, 1983:
Mean Nei’s D = 0.129). Moreover, genetic
distances are not appropriate measures of
taxonomic status for recently diverged union-
oid populations (e.g., Davis et al., 1981). “т
no case is the species concept based on ge-
netic distance alone” (Davis, 1983). A sim-
plistic reliance on genetic distance-based
taxonomic concepts should be abandoned.
Regarding genetic identity levels, SA (p.
366) states that “... Davis et al. (1981) ar-
gued that values > 0.9 could be found among
sympatric freshwater mussel species that re-
cently underwent speciation. However, these
two so-called subspecies of L. radiata are
currently not sympatric. . . .” This statement,
combined with SA's emphasis on genetic
distance, implies that allopatric populations,
in order to be recognized taxonomically,
must be more divergent allozymically than
sympatric populations. Can there not be dis-
tinct allopatric species with absolute genetic
identities greater than 0.9? Theoretically, any
number of assayed allozyme loci could indi-
cate genetic identity between two distinct
species. This may be expected for relatively
recently diverged taxa (e.g., Johnson et al.,
1977; Woodruff 4 Gould, 1980; Davis et al.,
1981; Carson, 1982; Kat, 1983). However,
genetic differences could still exist at non-
allozyme loci. How are these potential differ-
ences evaluated? They are evaluated by ref-
erence to morphological, ecological, and
phenological data. “The following species
concept is used here: a species of unionid is
a single lineage comprised of one or more
populations that diverge from other lineages.
Divergence is shown by significant morpho-
logical, cytological, reproductive biological
and/or ecological differences. . . . The case
for species status is strengthened if repro-
ductive isolation is highly probable due to
drainage system differences . . .” (Davis,
1983). We believe that the lack of reference
to genetic distance in this unionid species
concept is appropriate.
The concepts of (1) divergence (and, there-
fore, diagnosability) and (2) a lineage of pop-
ulations (both following Davis, 1983) should
be incorporated in evaluations of unionoid
taxonomic boundaries. The limited data
available in SA (tables 2 & 3, range and hab-
itat data provided in the text) suggest that
both Lampsilis r. conspicua and L. fullerkati
are diagnosable from L. r. radiata. Despite the
small number of loci assayed and minimal
genetic distances reported in SA, the allo-
zyme data set does suggest that there are
diagnostic alleles (i.e., those alleles found
in only one of the three taxa of interest here:
L. r. radiata, one diagnostic allele; L. r. con-
spicua, eight diagnostic alleles; and L. fuller-
kati, four diagnostic alleles). The presence of
unique genetic elements in a particular pop-
ulation is not consistent with a hypothesis of
current genetic interchange with the other
populations. Therefore, if these alleles remain
diagnostic after adequate sampling of popu-
lations of the three nominal taxa, this would
be strong evidence consonant with the dis-
tinctness of these populations. This sugges-
226 HOEH & GORDON
tion of distinctness is further evinced by the
morphological and ecological data presented
in SA.
CONCLUSIONS
The significant conchological, habitat, and
range differences reported in SA (e.g., both in
the text and table 2), suggest that the popu-
lations identified as L. fullerkati, L. r. radiata
and L. r. conspicua represent distinct evolu-
tionary lineages. Apparent non-identity of al-
lozymic composition for these populations
(SA: table 3) is consonant with this hypothe-
sis. We believe that SA's “suggestion . . . of
regrouping the two and possibly three previ-
ously recognized allopatric subspecies/spe-
cies into one species complex, based upon
very high levels of genetic identity as well as
similar conchologies, is probably an uncom-
mon event, yet appropriate until more dis-
tinctive biological species properties become
evident” (SA: 367) is not appropriate at this
time. Given the above discussion and the
rapid decline of North American unionoid
populations (e.g., Bogan, 1993), we believe
that it is a scientifically justifiable and conser-
vative course of action to continue to recog-
nize L. r. conspicua (sensu SA) and L. fuller-
kati as taxa distinct from L. r. radiata. Data
indicating need for further revision within the
L. radiata group may become available; how-
ever, any revisions should necessarily be
based on rigorous analyses of all the avail-
able characteristics of individuals selected by
appropriate sampling criteria.
ACKNOWLEDGMENTS
We thank А. E. Bogan, J. В. Burch, 5. L.
Neale, E. E. Spamer, and D. T. Stewart for
their criticisms of earlier versions of this com-
mentary. We also appreciate the comments
of an anonymous reviewer as well as those of
the editor, George M. Davis.
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MALACOLOGIA, 1996, 38(1-2): 229-230
A CALL FOR A NEW INTERNATIONAL CONGRESS OF ZOOLOGY
Dr. Е. D. Por! & Dr. В. М. Polymeni*
We are looking for response concerning
the feasibility of a New International Con-
gress of Zoology, possibly to be convened in
Athens, Greece, sometime during 1999 or
2000.
The First International Congress of Zool-
ogy was held in Paris, in 1889. Seventy years
later, the XVIth Congress in Washington rec-
ommended the discontinuation of the con-
gresses because of the feeling that zoology
had split into too many unrelated, specialized
fields. Nonetheless, a last XVIIth rump Con-
gress was held in 1972 in Monte Carlo. The
relatively few participants of this meeting
unanimously, but in vain, asked for the con-
tinuation of the congresses. The idea was ad-
vanced that the International Conferences on
Systematic and Evolutionary Biology would
replace the defunct Zoological Congresses
at a higher, integrative level. After several
meetings of the ICSEB, it became evident
that they did not live-up to this expectation.
In contrast, the International Congresses of
Botany have continued undisturbed and suc-
cessfully.
One of the unhappy consequences of the
cessation has been the fact that the Interna-
tional Commission of Zoological Nomencla-
ture, once accountable to the congresses,
came under the formal responsibility of the
General Congresses of International Union of
the Biological Sciences (IUBS). But the more
painful and long-lasting consequence was a
general depreciation of zoology in the aca-
demic world as such, and the replacement of
this discipline by a plethora of euphemistically
more fashionable designations.
However zoology at the end of this century
is more alive than ever and rich in new ideas
and achievements. A multispeciality ex-
change of views is more necessary than ever
before. Not unexpectedly, this is also the con-
sequence of the extreme parochialism of the
different splinter fields and the ignorance of
general zoological issues which it generated.
There is a long list of such issues that cut
across the lines of all the zoological special-
ities, some of them of important philosophical
and practical significance.
The widely circulated ‘“‘Systematics Agenda
2000” emphasizes our present incapacity to
describe scientifically a zoological biodiver-
sity that suddenly appears to be one order of
magnitude larger than envisaged in the 1970s.
This is not only a matter of quantities or of time
needed, but a matter that calls for the restruc-
ture of zoological research world over. A crit-
ically depleted and weakened community of
zoological systematics cannot live up to the
task to investigate and possibly protect the
heritage of the animal world.
On the positive side, there have been many
developments during the last three decades
that need to be appreciated by an interna-
tional forum of all the zoologists. Confined to
the pages of specialized journals, these im-
portant developments often do not reach the
attention of peers in other zoological special-
ties. In the field of more classical zoology, it
would be useful to acquaint our colleagues
with such topics as the recently discovered
new animal phyla and classes, new concepts
of vertebrate evolution, zoology of clonal
animals, and present views on the Protozoa.
A sample of subjects of wider implications
are sociobiology, cladistics, molecular taxon-
omy, modern embryology, the new vision on
the Cambrian revolution, the neo-catastro-
phism, vicariance zoogeography, in situ and
ex situ conservation, cryopreservation, and
cloning. This is a different zoology from that
which ended with a whimper in Monte Carlo.
We are ready to try to bring forward again
the rich and unifying aspects of zoology and
to reassert its general global, human and
philosophical role. We are hoping for the ap-
proval and support of the zoological dias-
pora. The best encouragement will be to
send us suggestions regarding the themes
and the structure of the proposed New Inter-
national Congress of Zoology. More impor-
tantly, we need personal commitments to
"Department of Evolution, Systematics and Ecology, Hebrew University of Jerusalem, POB 91904, Jerusalem, Israel
“Department of Biology, Section of Zoology, University of Athens, POB 15784, Athens, Greece.
Please contact Dr. Rosa Polymeni; Tel. 30.1.7284364, Fax 30.1.7284604, email: rpolime@atlas.uoa.gr
230 POR & POLYMENI
help organizing symposia, workshops and
hints of possible funding sources. We shall
also need to establish an active and repre-
sentative Action Committee. Understand-
ably, we shall be able to appeal for funding
only after having obtained convincing public
support and after having a prestigious
committee in place.
The editor-in-chief of Malacologia welcomes let-
ters that comment on vital issues of general im-
portance to the field of Malacology, or that com-
ment on the content of the journal. Publication is
dependent on discretion, space available and, in
some cases, review. Address letters to: Letter to
the Editor, Malacologia, care of the Department
of Malacology, Academy of Natural Sciences,
19th and the Parkway, Philadelphia, PA 19103.
MALACOLOGIA, 1996, 38(1-2): 231-239
INDEX
Page numbers in /ta/ics indicate
illustrations of taxa.
Acanthinula 165, 166, 171, 178
Achatina stuhlmanni 165, 166, 169,
171, 174, 179
Acochlidium amboinense 147
bayerfehlmanni 147, 150
fijiense 143-151; 144, 145, 147, 148
sutteri 147, 150
weberi 147
Actinonaias ligamentina 184, 186-189,
202
Adula californiensis chosenica 36, 40, 41
schmidti 41
Afroconulus diaphanus 180
iredalei 165, 166, 171, 179
agapetus, Potamolithus 1-17; 2, 9
agrestis, Agriolimax 156, 157
Agriolimax agrestis 156, 157
reticulatus 157
Alasmidonta marginata 184, 186, 187,
192,202
undulata 182
Albinaria coerulea 216-219
Alderja modesta 149
Allogona profunda 67-86; 70-73
aloysiisabaudiae, Gymnarion 165, 166,
171, 180
alveata, Lirophora 110
amathusia, Chionopsis 109, 112, 116-
124, 128-130, 142
Amblema plicata 182, 184, 186, 187,
189,195, 197, 201, 202
amboinense, Acochlidium 147
Ampelita 207
Ancistrolepis trochoidea ovoidea 36, 38
trochoidea [Bathyancistrolepis] ovoidea
38
trochoideus ovoideus 38
Anodonta imbelicis 59-65
Anodontoides ferussacianus 184, 187,
194, 202
Anomalocardia 108, 111, 113, 127, 133-
135, 140
auberiana 116-124, 142
flexuosa 112, 116-124, 142
Anomiostrea 36, 45
coralliophila 45
pyxidata 45
Appalachina sayana 67-86; 70-73
appressa, Patera 67-86
araneosa, Chione 109
ariel, Trachycystis 167, 179
armata, Bullia 96
armatum, Buccinum 96
armatum, Dorsanum 96
asperrima, Protothaca (Leucoma) 112
asperrima, Protothaca 112, 116-124,
142
aspersa, Helix 147
Astralium yamamurae 36, 41
yamamurai 41
Astralium (Destellifer) yamamurae 41
Astralium (Distellifer) yamamurae 41
athleta, Lirophora 110, 112, 116-124,
127,129, 142
auberiana, Anomalocardia 116-124, 142
auriformis, Daedalochila 67, 83
Australaba 56
babaulti, Curvella 165, 166, 169, 171,
179
bacillum, Streptostele 165, 166, 171,
180
bainbridgensis, Chione (Chione) 111
bainbridgensis, Puberella 110
ballista, Lirophora 110
barbatum, Stenotrema 67
barbigerum, Stenotrema 67-86; 80, 81
barrakporensis, Kaliella 165, 166, 171,
179
batillariaeformis, Clypeomorus 36, 42
batillariaeformis, Clypeomrus 42
bayerfehlmanni, Acochlidium 147, 150
beringii, Beringion 39
Beringion 36, 39
beringii 39
marshalli 39
bifasciata, Clypeomorus 47-58; 49, 50,
52, 54
Biomphalaria glabrata 59, 62, 63
bisulcata, Nesopupa 165, 166, 168, 169,
171, 178
Boreomelon stearnsii ryosukei 36, 40
Boreotrophon paucicostatus 36, 37
Boromelon stearnsii ryosukei 40
Bosellia corinneae 149
Boucardicus 207, 209, 210
Brachytoma kawamurai 36, 44
kurodai 36, 44
vexillium 36, 44
Bradybaenidae 85
breviculus, Clypeomorus 56
brunnea, Eglisia 36, 44, 45
brunnea, Eglisia lanceolata 45
Buccimum chishimana nux 38
Buccinanops cochlidium 96
cochlidius 87
deformis 87
gradatus 87-102; 89, 90, 92-96
lamarcki 87
moniliferus 87-102; 89, 98-100
monliferum 96
231
232 INDEX
uruguayensis 87
buccinoides, Clinopegma 36, 38, 39
Buccinum armatum 96
chishimananux 35, 38
chishimanum nux 36, 38
felis shikamai 36, 38
ferrugineum 36, 39, 40
hosoyal 36, 38
japonicum 38
kawamurai 36, 38
kinukatsugi 36, 39, 40
midori 36, 40
moniliferum 96
opisthoplectum microconcha 36, 38
subreticulatum 36, 39
undatum 101
Buccinum (Buccinanops) maniliferum 96
Bulbus flavus elongatus 36, 37
Bullia 87
armata 96
Bullia (Buccinanops) moniliferum 96
burnsil, Panchione 110
buschii, Lithoglyphus 7
bushchii, Potamolithus 1-17; 10-12, 14-
16
butumbiana, Prositala 165, 166, 171,
179
caeruleum, Cerithium 56
californiensis, Chione 109, 130
Camaenidae 85
canarium, Strombus 3
cancellata, Chione 109, 112, 116-124,
127. 129; 130. 132, 135; 142
cancellata, Harpa 44
cardium, Lampsilis 184, 186-191, 202
carinifer, Hemifusus 36, 43
carinifera, Hemifusus 43
cariosa, Lampsilis 223, 224
carlottae, Lirophora 110
caroniana, Lirophora 110
castanea, Neptunea 39
castaneum, Volutopsion 39
castaneus, Volutopsion 39
castaneus, Volutopsius 39
cataracta, Pyganodon 182
Cecilioides 164-166
Cerastua trapezoidea lagariensis 165,
166, 169, 171, 178
Сераеа nemoralis 216-219
Cerithium 56
caeruleum 56
nodulosum 56
rupestre 56
vulgatum 56
Chione 103-142
araneosa 109
californiensis 109, 130
cancellata 109, 112, 116-124, 127,
129, 130, 132, 185; 142
chipolana 109, 111, 112, 116-124,
132, 142
compta 109
erosa NOS 129130
guatulcoensis 109
mazyckii 109, 129
pallasana 109
primigenia 109, 127
quebradillensis 109
raca 129
santodomingensis 109, 127
subimbricata 109, 111, 125
tumens 109, 112, 113, 116-125 723:
130
undatella 106, 109, 129
vaca 125, 128
Chione (Chione) bainbridgensis 111
spenceri 111
subimbricata 111
tumens 111
Chioninae 108
Chionista 108, 113, 125, 133, 1944157
cortezi 109, 129, 130
eurylopas 109
fluctifraga 109, 112, 116-124, 129,
130, 142
gnidia 109
jamaniana 109
ornatissima 109
posorjensis 109
procancellata 109
propinqua 109
rowleei 110
tegulum 110
Chionopsis 108, 111, 113, 126; 131%
132-134, 137, 140
amathusia 109, 112, 116-124, 128-
130, 142
gnidia 129,130, 136
lliochione 110
procancellata 127,129,130
subrugosa 110
tegulum 127,130, 136
wally 110
woodwardi 110, 127, 129, 130
chipolana, Chione 109, 111, 112, 116-
124, 132, 142
chiriquiensis, Lirophora 110
chishimananum, Buccinum 35, 38
chiui, Oncomelania hupensis 213
Chlamydarion oscitans 165, 166, 169,
171,180
Chorus giganteus 55
chosenica, Adula californiensis 36, 40,
41
cinnamomeozonata, Thapsia 180
Cirsotrema kagayai 36, 37
clara, Subulona 165, 166, 169, 171, 178
clava, Pleurobema 182,184, 186, 187,
192, 193, 202
Clavator 207
clenchi, Lirophora 110
Clinopegma buccinoides 36, 38, 39
Clypeomorus batillariaeformis 36, 42
INDEX
bifasciata 47-58; 49, 50, 52, 54
breviculus 56
moniliferum 48
moniliferus 56
petrosa gennesi 48
tuberculatus 47-58; 50, 52, 54
Clypeomrus batillariaeformis 42
coccineum, Pleurobema 186
cochlidium, Buccinanops 96
cochlidius, Buccinanops 87
coerulea, Albinaria 216-219
Collisella cassis shirogai 35, 37
pelta shirogal 35, 37
colombiana, Crenella 40
colombiana, Megacrenella 40
columbiana, Vespericola 67-86
complanata, Lasmigona 184, 187, 194,
195, 202
compressa, Lasmigona 184, 187, 194,
202
compta, Chione 109
Conomurex 56
Conulinus 174
rutshuruensis major 165, 166, 169,
171: 178
conspicua, Lampsilis radiata 223-226
coralliophila, Anomiostrea 45
Corbicula fluminea 20
corinneae, Bosellia 149
cornuta, Pseudunela 147, 149
cortezi, Chionista 109, 129, 130
cortinaria, Puberella 110, 127
costata, Lasmigona 182, 184, 186, 187,
191, 194, 202
Crenella colombiana 40
cribaria, Puberella 110, 112, 116-124,
129, 130, 142
Curvella 165, 166, 171, 179
babaultí 165, 166, 169, 171,179
Cyathopoma 207, 210
cylindrica, Quadrula 184,186, 187, 202
Cypraea pulchella 42
Daedalochila auriformis 67,83
dalli, Lirophora 110
dalli, Nodulotrophon 37
dalli, Trophon 38
Decollidrillia 36, 40
nigra 36, 40
deformis, Buccinanops 87
denotata, Xolotrema 67-86; 77
densesculpta, Thapsia 180
Dentalium (Pictodentalium) formosum 34
formosum hirasei 34
dentifera, Neohelix 67-86; 76
Deroceros reticulatus 157
diaphanus, Afroconulus 180
dilatata, Elliptio 184, 186, 187, 189,
192, 202
discrepans, Lirophora 110
disseminata, Gulella 165, 166, 170, 171,
180
233
doerfeuilliana, Millerelix 67
Dorsanum 87
armatum 96
miran 87, 101
moniliferum 96
Dreissena polymorpha 19-31
dubia, Palio 149
ebergenyi, Lirophora 110
edulis, Mytilus 28,218
Eglisia brunnea 36, 44, 45
lanceolata brunnea 45
elatior, Maizania 165, 166, 168, 169,
1717178
elegans, Pseudoglessula (Ischnoglessula)
179
elegans, Pseudoglessula 165, 166, 171,
172
elegans, Tantulum 143
Elgonocyclus koptaweliensis 165, 166,
171, 178
Elliptio dilatata 184, 186, 187, 189, 192,
202
elongatus, Bulbus flavus 36, 37
elongensis, Gonaxis 165, 166, 171, 180
elongensis, Thapsia 180
Elysia maoria 149
subornata 149
emphatica, Trophonopsis scitulus 37
emphaticus, Trophonopsis scitula 36, 37
emphaticus, Trophonopsis scitulus 37
Epioblasma 186
torulosa 184, 202
torulosa rangiana 182, 187-191
triquetra 184, 186-191, 202
erosa, Chione 109, 129, 130
eucosmia, Thapsia 164-166, 168, 169,
180
euracantha, Murex 42
euracanthus, Murex 42
eurantha, Murex 42
eurantha, Spinidrupa 42
eurylopas, Chionista 109
exquisita, Fusipagoda 38
exquisita, Mohnia 38
fabalis, Villosa 184, 186-188, 191, 192,
202
falconensis, Lirophora 110
fallax, Triodopsis 67-86; 70-73
fasciola, Lampsilis 184, 186-190, 202
fasciolaris, Ptychobranchus 184, 186-
189, 192, 198, 202
Fauxulus 207, 210
ferrissi, Inflectarius 67-86; 76, 81
ferrugineum, Buccinum 36, 39, 40
ferussacianus, Anodontoides 184, 187,
194, 202
Ficadusta 36, 42
Ficadusta pulchella 42
fijense, Acochlidium 143-151; 144, 145,
147, 148
234
flexuosa, Anomalocardia 112, 116-124,
142
fluctifraga, Chionista 109, 112, 116-124,
1292 130, 142
fluminea, Corbicula 20
formosana, Oncomelania hupensis 213
formosum, Dentalium (Pictodentalium) 34
fosteri, Xolotrema 67-86; 70-73
frielei, Neoberingus 39
Fulgoraria (Musashia) kaneko hayashii 36,
40
fullerkati, Lampsilis 223-226
funiakensis, Panchione 110
Fusconaia subrotunda 184, 186, 187,
192, 193; 202
Fusinus 33
hyphalus 33
Fusinus (Simplicifusus) hyphalus 33
Fusipagoda 36, 38
exquisita 38
Fusus simplex 33
gennesi, Clypeomorus petrosa 48
gerstenbrandti, Thapsia 180
gibbosula, Heterocardia 45
gibbosuloidea, Plicarularia 36, 43
giganteus, Chorus 55
glabrata, Biomphalaria 59, 62, 63
gnidia, Chionista 109
gnidia, Chionopsis 129, 130, 136
Gonaxis elongensis 165, 166, 171, 180
gradatus, Buccinanops 87-102; 89, 90,
92-906
grandis, Pyganodon
195,202
Granulittorina 36, 41, 42
millegrana 42
Granulittorina philippiana 36, 41, 42
guatulcoensis, Chione 109
Gulella disseminata 165, 166, 170, TALA
180
disseminata var. kekumegaensis 180
handeiensis 165, 166, 171, 180
impedita 165-167, 170, 171, 180
lessensis 165, 166, 171, 180
osborni 165, 166, 170, 171, 180
184, 187, 189, 194,
ugandensis 165-167, 169, 171, 180
woodhousei 165, 166, 171, 180
Gulella (sect. Silvigulella) 174
Guppya quadrisculpta 165, 166, 169,
19; 179
Gymnarion aloysiisabaudiae 165, 166,
171; 180
Hainesia 207
Halolimnohelix percivali 165, 166, 171
plana 165, 166, 171, 180
handeiensis, Gulella 165, 166, 171, 180
Harpa cancellata 44
harpa 44
Калуата! 36, 44
kawamurai 36, 43, 44
INDEX
major 44
striata 43
harpa, Harpa 44
Harpofusus 35,36, 39
melonis 39
harpula, Pupisoma (Salpingoma) 178
harpula, Pupisoma 165, 166, 171
hayashii, Fulgoraria (Musashia) kaneko
36, 40
Hedylopsis 149
spiculifera 148, 149
Helicidae 85
Helicophanta 207
Helix aspersa 147
Hemifusus carinifer 36, 43
carinifera 43
hendersoni, Lirophora 110, 129, 130
Heterocardia gibbosula 45
hirasei, Dentalium (Pictodentalium)
formosum 34
holocyma, Panchione 110
hosoyai, Buccinum 36, 38
hotelensis, Panchione 110
hupensis, Oncomelania 213-218, 220
hupensis, Oncomelania hupensis 213
hyphalus, Fusinus (Simplicifusus) 33
hyphalus, Fusinus 33
lliochione 108, 113, 125, 133, 134, 137,
140
subrugosa 112, 116-124, 129, 130,
142
imbelicis, Anodonta 59-65
impedita, Gulella 165-167, 170, 171,
180
Inflectarius ferrissi 67-86; 76, 81
inflectarius 68
inflectus 67-86; 70-73, 81
magazinensis 67-86; 80
smithi 67-86; 80
subpalliatus 67-86; 80
inflectarius, Inflectarius 68
inflectus, Inflectarius 67-86; 70-73, 81
intapurpurea, Puberella 111, 129
iredalei, Afroconulus 165, 166, 1741701879
iredalei, Kaliella 165, 166, 171, 179
iredalei, Oreohomorus 165, 166, 171,
179
iredalei, Trachycystis 165, 166, 171
iris, Villosa 184, 186-191, 202
isabella, Laevistrombus 33
Isabella, Laevistrombus canarium "forma"
33
jamaniana, Chionista 109
japonicum, Buccinum 38
japonicum, Pupisoma 178
kagayai, Cirsotrema 36, 37
Kajiyamai, Harpa 36, 44
Kalidos 207, 210
Kaliella 207, 210
INDEX 235
barrakporensis 165, 166, 171, 179
iredalei 165, 166, 171, 179
karamwegasensis, Thapsia 180
Katharina tunicata 218
kawamurai, Brachytoma 36, 44
kawamurai, Buccinum 36, 38
kawamurai, Harpa 36, 43, 44
kekumegaensis, Gulella disseminata var.
180
kekumeganum, Pseudopeas 179
kelletii, Panchione 110, 112
kinukatsugi, Buccinum 36, 39, 40
koptawellilensis, Micractaeon 165, 166,
WORDT, 178
koptaweliensis, Elgonocyclus 165, 166,
ТУТ, 178
koreana, Megacardita ferruginea 41
koreana, Megacardıta furriginosa 36, 41
koreanica, Megacardita ferruginea 41
kurodai, Brachytoma 36, 44
kuroshio, Neptunea 35
lacteoides, Pyrene 36, 43
Laevicardium rubropictum 36, 45
laevior, Patera 67-86; 70-73
Laevistrombus 33
canarium "forma" /sabella 33
Isabella 33
lagariensis, Cerastua trapezoidea 165,
166: 169: 1711783
lamarckii, Buccinanops 87
Lambis 56
Lampsilis cardium 184, 186-191, 202
cariosa 223, 224
fasciola 184, 186-190, 202
fullerkati 223-226
ovata 184, 186-191, 202
radiata 223
radiata conspicua 223-226
radiata radiata 223-226
siliquoidea 182, 184, 186-189, 191,
196, 201, 202
splendida 223
lapidum, Potamolithus 1
Lasmigona complanata 184,187, 194,
195; :202
compressa 184, 187, 194, 202
costata 182, 184, 186, 187, 191,
194, 202
latilirata, Lirophora 110, 129
Latirus recurvirostrum 43
stenomphalus 36, 43
sttnomphalus 43
Lehmania valentiana 157
Leptodea ochracea 223
lessensis, Gulella 165, 166, 171, 180
Leukoma 108, 140
ligamentina, Actinonaias 184, 186-189,
202
Ligumia nasuta 184, 186, 187, 189,
195, 202
recta 184, 186-191, 202
Limaria perfragile 45
Limaria (Platilimaria) perfragile 45
Limax maximus 153-160; 155, 159
Limicolaria saturata 165-167, 170, 171,
179
lindoensis, Oncomelania hupensis 213
Lirophora 108, 111, 113, 126, 127, 130.
131, 133-135, 137 140
alveata 110
athleta 110, 112, 116-124, 127, 129,
142
ballista 110
carlottae 110
caroniana 110
chiriquiensis 110
clenchi 110
Ча! 110
discrepans 110
ebergenyi 110
falconensis 110
hendersoni 110, 129, 130
latilirata 110, 129
mariae 110
obliterata 110
paphia 110, 129, 130
quirosensis 110
riomaturensis 110
sellardsi 110
tembla 110
victoria 110, 112, 116-124, 142
vrendenbergi 110
Lithoglyphus buschii 7
lliochione, Chionopsis 110
Lymnaea stagnalis 59, 62-64
Macrotoma yamamurae 36, 45
mactropsis, Panchione 110,112, 116-
124, 127, 136, 142
magazinensis, Inflectarius 67-86; 80
Maizania elatior 165, 166, 168, 169,
171,178
major, Conulinus rutshuruensis 165, 166,
169, 171; 178
major, Harpa 44
major, Neohelix 67-86; 71-73
Malagarion 207, 210
Malarinia 207, 210
maniliferum, Buccinum (Buccinanops) 96
Mantellum perfragile 36, 45
maoria, Elysia 149
Margarites vorticifera 37
marginata, Alasmidonta 184, 186, 187,
192.202
mariae, Lirophora 110
marica, Timoclea (Glycydonta) 112
marica, Timoclea 112,116-124, 142
marshalli, Beringion 39
maxillatum, Stenotrema 67-86; 80
maximus, Limax 153-160; 755, 159
mazyckii, Chione 109, 129
236
mcmichaeli, Volutoconus grossi 36, 44
medjensis, Trochozonites (Zonitotrochus)
180
medjensis, Trochozonites 165, 166, 171
Megacardita ferruginea koreana 41
ferruginea koreanica 41
ferruginosa koreana 36, 41
Megacrenella 35, 36, 40
Megacrenella colombiana 40
Melanopsis 55
melonis, Harpofusus 39
melonis, Pyrulofusus (Harpofusus) 39
melonis, Strombella 39
Mercenaria 113, 132-134, 140
mercenaria 112, 116-124, 142
mercenaria, Mercenaria 112, 116-124,
142
Mesodon normalis 67-86; 70, 72, 73
Mesodontini 83
Micractaeon koptawellilensis 165, 166,
170,171; 178
microconcha, Buccinum opisthoplectum
36, 38
Microcystis 207, 209, 210
microleuca, Thapsia 164-166, 180
Microtoma yamamurae 45
midori, Buccinum 36, 40
millegrana, Granulittorina 42
millegrana, Nodilittorina (Granulittorina)
42
Millerelix doerfeuilliana 67
doerfeuilliana sampsoni 67
mooreana 67
mime, Thapsia 180
minima, Oncomelania 213
miran, Dorsanum 87, 101
modesta, Alderja 149
Mohnia exquisita 38
multicostata 36, 38
moniliferum, Buccinum 96
moniliferum, Bullia (Buccinanops) 96
moniliferum, Clypeomorus 48
moniliferum, Dorsanum 96
moniliferus, Buccinanops 87-102; 89, 98-
100
moniliferus, Clypeomorus 56
monliferum, Buccinanops 96
montezuma, Puberella 111
mooreana, Millerelix 67
morsitans, Puberella 111, 129, 130
multicostata, Mohnia 36, 38
Murex euracantha 42
euracanthus 42
eurantha 42
mutandana, Pseudoglessula
Mytilus edulis 28, 218
179
Nassarius reticulata 101
nasuta, Ligumia 184, 186, 187, 189,
195, 202
Nebularia yaekoae 36, 43
INDEX
nebulosa, Villosa 182
nemoralis, Cepaea 216-219
Neoberingus 36, 39
frielei 39
Neohelix dentifera 67-86; 76
major 67-86; 71-73
Neptunea 35
castanea 39
kuroshio 35
rurosio 35
Nesopupa bisulcata 165, 166, 168, 169,
MIRAS
nigra, Decollidrillia 36, 40
nigropardalis, Pyrene testudinalia 42, 43
nigropardalis, Pyrene testudinaria 36, 42,
nigropunctatum, Vasticardium 36, 45
ninagongonis, Truncatellina 165, 166,
1171541478
nitidus, Oreohomorus 179
Nodilittorina 42
Nodilittorina (Granulittorina) millegrana 42
nodulosum, Cerithium 56
Nodulotrophon 36-38
dalli 37
normalis, Mesodon 67-86; 70, 72, 73
nosophora, Oncomelania hupensis 213
Nothapalus 165, 166, 171, 174, 178
nux, Buccimum chishimana 38
nux, Buccinum chishimanum 36, 38
obliterata, Lirophora 110
obstricta, Xolotrema 67-86; 77, 78
ochracea, Leptodea 223
olssoni, Puberella 111
Omphalomargarites 36, 37
vorticifera 207, 210
Omphalomargarites (Omphalomargarites)
37
Oncomelania 213-221
hupensis 213-218, 220
hupensis chiui 213
hupensis formosana 213
hupensis hupensis 213
hupensis lindoensis 213
hupensis nosophora 213
hupensis quadrasi 213
hupensis robertsoni 213, 218
hupensis tangi 213
minima 213
orcula, Pupisoma 165, 166, 171, 178
Oreohomorus 174
iredalei 165, 166, 171, 179
nitidus 179
ornatissima, Chionista 109
osborni, Gulella 165, 166, 170, 171, 180
oscitans, Chlamydarion 165, 166, 169,
171, 180
Ostrea pyxidata 45
ovata, Lampsilis 184, 186-191, 202
ovata, Timoclea 112
INDEX
ovoidea, Ancistrolepis trochoidea 36, 38
ovoidea, Ancistrolepis trochoidea
[Bathyancistrolepis] 38
ovoideus, Ancistrolepis trochoideus 38
pallasana, Chione 109
Palio dubia 149
zosterae 149
Panchione 108, 113,126, 127, 131,
125485187140
burnsii 110
funiakensis 110
holocyma 110
hotelensis 110
kelletii 110, 112
mactropsis 110, 112, 116-124, 127,
136, 142
parkeria 110
ulocyma 112, 116-124, 127, 129,
130, 142
paphia, Lirophora
paradoxa, Strubellia 143, 149
parkeria, Panchione 110
Patelloida (Collisellina) saccharinoides 36,
41
saccharioides 41
Patera appressa 67-86
appressa sculptior 78, 79
laevior 67-86; 70-73
perigrapta 67-86; 78
sargentiana 67-86; 78
paucicostatus, Boreotrophon 36, 37
percivali, Halolimnohelix 165, 166, 171
perfragile, Limaria (Platilimaria) 45
perfragile, Limaria 45
perfragile, Mantellum 36, 45
perigrapta, Patera 67-86; 78
Petenopsis 140
tumens 142
pfeifferianus, Reticutriton 42
philippiana, Granulittorina 36, 41, 42
Pictodentalium 34
pilosa, Vespericola columbiana 67-86; 80
plana, Halolimnohelix 165, 166, 171,
180
planulata, Trachycystis 172
Pleurobema clava 182, 184, 136, 187,
192,193. 202
coccineur.. 186
sintoxia 184, 186, 187, 189, 192,
193,195, 201,202
Plicarularia gibbosuloidea 36, 43
plicata, Amblema 182, 184, 186, 187,
189, 195, 197, 201, 202
Polygyridae 83
polymorpha, Dreissena 19-31
Pomatiopsinae 220
posorjensis, Chionista 109
Potamolithus agapetus 1-17; 2, 9
bushchii 1-17; 10-12, 14-16
lapidum 1
primigenia, Chione 109, 127
1107, 129,130
237
procancellata, Chionista 109
procancellata, Chionopsis 127, 129, 130
profunda, Allogona 67-86; 70-73
propinqua, Chionista 109
Prositala 174
butumbiana 165, 166, 171, 179
Protothaca 108, 113, 132-134
asperrima 112, 116-124, 142
Protothaca (Leucoma) asperrima 112
Pseudoglessula elegans 165, 166, 171,
172
mutandana 179
subfuscidula 179
Pseudoglessula (Ischnoglessula)
elegans 179
Pseudopeas kekumeganum 179
yalaensis 165, 166, 171, 172, 179
Pseudunela cornuta 147, 149
Ptychobranchus fasciolaris 184, 186-
189192195820?
pubera, Puberella 111
Puberella 108, 111, 113, 126, 131, 132-
134, 137, 140, 141
bainbridgensis 110
cortinaria 110,127
cribaria 110, 112, 116-124, 129, 130,
142
intapurpurea 111,129
montezuma 111
morsitans 111, 129, 130
olsson 111
pubera 111
pulicaria 111, 129, 130
purpurissata 111
sawkinsi 111, 127, 129, 130
pulchella, Cypraea 42
pulchella, Ficadusta 42
pulicaria, Puberella 111, 129, 130
Punctum ugandanum 165, 166, 169,
170; 174,179
Pupisoma harpula
japonicum 178
öreula 165, 166, 171,178
Pupisoma (Salpingoma) harpula 178
purpurissata, Puberella 111
Pyganodon cataracta 182
grandis 184, 187, 189, 194, 195, 202
Pyrene lacteoides 36, 43
testudinalia nigropardalis 42, 43
testudinaria nigropardalis 36, 42, 43
Pyrulofusus 39
Pyrulofusus (Harpofusus) melonis 39
pyxidata, Anomiostrea 45
pyxidata, Ostrea 45
174
165, 166,171
quadrasi, Oncomelania hupensis 213
quadrisculpta, Guppya 165, 166, 169,
171; 179
Quadrula cylindrica
quebradillensis, Chione
quirosensis, Lirophora
184, 186, 187, 202
109
110
238
raca, Chione 129
radiata, Lampsilis 223
radiata, Lampsilis radiata 223-226
rangiana, Epioblasma torulosa 182, 187-
91
recta, Ligumia 184, 186-191, 202
recurvirostrum, Latirus 43
reticulata, Nassarius 101
reticulatus, Agriolimax 157
reticulatus, Deroceros 157
Reticutriton 36, 42
pfeifferianus 42
Rhachidina chiradzuluensis var. virginea
165-167, 171, 178
Rhinoclavis 56
riomaturensis, Lirophora 110
robertsoni, Oncomelania hupensis 213,
218
rowleei, Chionista 110
rubocostatum, Vexillum 36, 43
rubropictum, Laevicardium 36, 45
rupestre, Cerithium 56
rurosio, Neptunea 35
ryosukei, Boreomelon stearnsii 36, 40
ryosukei, Boromelon stearnsii 40
saccharinoides, Patelloida (Collisellina)
36, 41
saccharioides, Patelloida (Collisellina) 41
sampsoni, Millerelix doerfeuilliana 67
santodomingensis, Chione 109, 127
sargentiana, Patera 67-86; 78
saturata, Limicolaria 165-167, 170, 171,
179
sawkinsi, Puberella 111, 127, 129, 130
sayana, Appalachina 67-86; 70-73
schmidti, Adula 41
sculptior, Patera appressa 78, 79
sellardsi, Lirophora 110
shikamai, Buccinum felis 36, 38
shirogai, Collisella cassis 35, 37
shirogai, Collisella pelta 35, 37
siliquoidea, Lampsilis 182, 184, 186-189,
191, 196, 201, 202
simplex, Fusus 33
Simplicifusus 33, 34
sintoxia, Pleurobema 184, 186, 187,
139, 192. 193, 195, 2017202
Sitala 207, 209,210
smithi, Inflectarius 67-86; 80
spenceri, Chione (Chione) 111
spiculifera, Hedylopsis 148, 149
Spinidrupa 36, 42
eurantha 42
splendida, Lampsilis 223
stagnalis, Lymnaea 59, 62-64
stenomphalus, Latirus 36, 43
Stenotrema barbatum 67
barbigerum 67-86; 80, 81
maxillatum 67-86; 80
Streptostele bacillum 165, 166, 171, 180
striata, Harpa 43
INDEX
Strombella melonis 39
Strombus canarium 3
Strophitus undulatus 184, 186, 187,
189, 192, 202
Strubellia paradoxa 143, 149
sttnomphalus, Latirus 43
stuhlmanni, Achatina 165, 166, 169,
le MAA AES
subcylindrica, Truncatella 55
subfuscidula, Pseudoglessula 179
subimbricata, Chione (Chione) 111
subimbricata, Chione 109, 111, 125
subornata, Elysia 149
subpall'atus, Inflectarius 67-86; 80
subreticulatum, Buccinum 36, 39
subrotunda, Fusconala 184, 186, 187,
19271937202
subrugosa, Chionopsis 110
subrugosa, lliochione 112, 116-124, 129,
130, 142
Subulona clara 165, 166, 169, 171, 178
Succinea 165, 166, 171, 178
sutteri, Acochlidium 147, 150
tangi, Oncomelania hupensis 213
Tantulum elegans 143
tegulum, Chionista 110
tegulum, Chionopsis 127, 130, 136
tembla, Lirophora 110
Thapsia cinnamomeozonata
densesculpta 180
elongensis 180
eucosmia 164-166, 168, 169, 180
gerstenbrandti 180
karamwegasensis 180
microleuca 164-166, 180
mime 180
yalaensis 180
Timoclea 108, 111, 113, 132-134, 140
marica 112, 116-124, 142
ovata 112
Timoclea (Glycydonta) marica 112
torulosa, Epioblasma 184, 202
Trachycystis ariel 167, 179
iredalei 165, 166, 171
planulata 172
Tricula 217,220
Triculinae 220
Triodopsini 83
Triodopsis fallax 67-86; 70-73
triquetra, Epioblasma 184, 186-191, 202
Trochozonites medjensis 165, 166, 171
Trochozonites (Zonitotrochus) 174
medjensis 180
Trophon dalli 38
Trophonopsis scitula emphaticus 36, 37
scitulus emphatica 37
scitulus emphaticus 37
Tropidophora 207, 210
Truncatella subcylindrica 55
Truncatellina ninagongonis 165, 166,
ПУ 78
180
INDEX
tuberculatus, Clypeomorus 47-58; 50,
52, 54
tumens, Chione (Chione) 111
tumens, Chione 109, 112, 113, 116-
125, 129, 130
tumens, Petenopsis 142
tunicata, Katharina 218
ugandanum, Punctum 165, 166, 169,
ТО 171 179
ugandensis, Gulella 165-167, 169, 171,
180
ulocyma, Panchione
129,130, 142
undatella, Chione 106, 109, 129
undatum, Buccinum 101
undulata, Alasmidonta 182
undulatus, Strophitus 184, 186, 187,
189, 192, 202
Unionoida 224
uruguayensis, Buccinanops 87
112 116-1247 127,
vaca, Chione 125, 128
valentiana, Lehmania 157
vanuxemensis, Villosa vanuxemensis 182
vanuxemi, Villosa 182
Vasticardium nigropunctatum 36, 45
Vespericola columbiana 67-86
columbiana pilosa 67-86; 80
vexillium, Brachytoma 36, 44
Vexillum rubocostatum 36, 43
victoria, Lirophora 110, 112, 116-124,
142
Villosa fabalis 184, 186-188, 191, 192,
202
iris 184, 186-191, 202
nebulosa 182
vanuxemensis vanuxemensis 182
vanuxemi 182
virginea, Rhachidina chiradzuluensis var.
165-167,171, 178
Volutoconus grossi mcmichaeli 36, 44
Volutopsion 36, 39
castaneum 39
castaneus 39
Volutopsius castaneus 39
vorticifera, Margarites 37
vorticifera, Omphalomargarites 207, 210
vrendenbergi, Lirophora 110
vulgatum, Cerithium 56
walli, Chionopsis 110
weberi, Acochlidium 147
woodhousei, Gulella 165, 166, 171, 180
woodwardi, Chionopsis 110, 127, 129,
130
Xolotrema denotata 67-86; 77
fosteri 67-86; 70-73
obstricta 67-86; 77, 78
yaekoae, Nebularia 36, 43
239
yalaensis, Pseudopeas 165, 166, 171,
172, 179
yalaensis, Thapsia 180
yamamurae, Astralium (Destellifer) 41
yamamurae, Astralium (Distellifer) 41
yamamurae, Astralium 36, 41
yamamurae, Macrotoma 36, 45
yamamurae, Microtoma 45
уататига!, Astralium 41
zosterae, Palio 149
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VOL. 38, NO. 1-2 MALACOLOGIA | rine 19988
CONTENTS | “O
MARÍA FERNANDA LÓPEZ ARMENGOL +0
Taxonomic Revision of Potamolithus ме. Pilsbry, 1911, ane Potamolithus de }
Buschii (Frauenfeld, 1865) (Gastropoda: KHydrobiidae) ea PRE ER Е
М. Е. CHASE & В. С. BAILEY | on
Recruitment of Dreissena Polymorpha: Does the Presence and Density of Conspe- _ dl
cifics Determine the Recruitment Density and Pattern in a Population? . LN
RUDIGER BIELER 8 RICHARD E. PETIT - = Уи
Additional Notes on Nomina First Introduced by Tetsuaki Kira in “Coloured Illustra- >
tions oùthe Shells of Japan 25". LCL ash. ala fe LAURE Е Re
RICHARD E. PETIT & RÜDIGER BIELER | \
3 On The New.Names Introduced in the Various Printings of “Shells of the World in + .
Colour” [Vol. | by Tadashige Habe and Kiyoshi Ito; Vol. Il by Tadashige Habe ne
Sadao Kosugel, ar IN courte Seat ae ails. О о. dood x 12 38
FADWA A. ATTIGA & HAMEED A. AL-HAJJ YA
Ultrastructural Study of Euspermiogenesis in Clypeomorus BES and Clypeo-
morus Tuberculatus (Prosobranchia: Cerithiidae) With Emphasis on Acrosome For-
MAHON: caes a a as da SA
DAZHONG XU & MICHELE G. WHEATLY ;
CA Regulation in the Freshwater Bivalve Anodonta Imbecilis: |. Effect of Environmen-
tal CA Concentration’and Body Mass on Unidirectional and Net CA Fluxes ......
KENNETH C. EMBERTON POSTE
Microsculptures of Convergent and Divergent Polygyrid Land-Snail Shells ........
LUIZ RICARDO L. SIMONE
Anatomy and ‘Systematics of Buccinanops Gradatus (Deshayes, 1844) and Bucci-
nanops Moniliferus (Kiener, 1834) SRE Muricoidea) From the Southeast-
em Coast of Brazil Run". di pp ME RON EEE CAS
PETER D. ROOPNARINE
Systematics, Biogeography and Extinction of Chionine Bivalves (Bivalvia: Veneridae)
in Tropical America: Early Oligocene-Recent .:.1.#..1..,.,,1.....,.L.4R scenes
MARTIN HAASE & ERHARD WAWRA
The Genital System of Acochlidium fijiense (Opisthobranchia: Acochlidioidea).and its
Infemed: РОСНО as RER OC SR ha ee I a) Re
G. M. KUCHENMEISTER, D. J. PRIOR & I. G. WELSFORD | Г
Quantification of the Development of the Cephalic Sac and Podocyst т the Тетез-
trial (Gastropod'Limax.Maximus LS M e ee г.
Р. TATTERSFIELD
Local Patterns of Land Snail Diversity т а Kenyan Rain Forest................. ES
LAURA R. WHITE, BRUCE A. MCPHERON, 8 JAY R. STAUFFER, JR.
Molecular Genetic Identification Tools for the Unionids of French Creek, Pennsylva-
—
|
KENNETH С. EMBERTON, TIMOTHY A: PEARCE & ROGER RANDALANA
Quantitatively Sampling Land-Snail Species Richness in Madagascan Rainforests .
CHRISTINA M. SPOLSKY, GEORGE M. DAVIS & ZHANG YI 4
Sequencing Methodology and Phylogenetic Analysis: Cytochrome b Gene Sequence
Reveals Significant Diversity in Chinese Populations of Oncomelania (Gastropoda:
Pomatiopsidae) ол ое PRESS | 21314
LETTERS TO THE EDITOR
WALTER В. HOEH & MARK E. GORDON
Criteria for the Determination of Taxonomic Boundaries in Freshwater Unionoids
(Bivalvia: Unionoida): Comments on Stiven and Alderman (1992) ................ 223 w
DR. F. D. POR & DR. R. M. POLYMENI Г
А Са! for a New International Congress of Zoology .............:::..:...... ed 229
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